Chimpanzee adenovirus vaccine carriers

ABSTRACT

The present invention provides recombinant replication-defective adenoviral vectors derived from chimpanzee adenoviruses and methods for generating recombinant adenoviruses in human E1-expressing cell lines. The invention also provides compositions and methods suitable for use for the delivery and expression of transgenes encoding immunogens against which a boosted immune response is desired. The invention further provides methods of generating clinical grade vector stocks suitable for use in humans. In a particular embodiment the invention contemplates the use of vectors comprising transgenes which encode tumor associated antigens in vaccines and pharmaceutical compositions for the prevention and treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application of U.S. Ser. No.10/587,389, filed Jul. 24, 2006, now U.S. Pat. No. 8,216,834, which is a§371 National Stage Application of PCT/EP2005/000558, internationalfiling date of Jan. 18, 2005, which claims the benefit of U.S.Provisional Application No. 60/538,799, filed Jan. 23, 2004, nowexpired, herein incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “ITR0048USDIV_SEQLIST 21JUN2012.TXT”, creation date of Jun.19, 2012, and a size of 653 KB. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of recombinant vectors andmore specifically to the production and use of recombinantreplication-defective chimpanzee adenoviral vectors to elicit immuneresponses in mammalian hosts.

BACKGROUND OF THE INVENTION

The adenoviruses (Ads) comprise a large family of double-stranded DNAviruses found in amphibians, avians, and mammals which have anonenveloped icosahedral capsid structure (Straus, Adenovirus infectionsin humans. In The Adenoviruses. 451-498, 1984; Hierholzer et al., J.Infect. Dis., 158: 804-813, 1988; Schnurr and Dondero, Intervirology.,36: 79-83, 1993; Jong et al., J Clin Microbiol., 37:3940-3945:1999). Incontrast to retroviruses, adenoviruses can transduce numerous cell typesof several mammalian species, including both dividing and nondividingcells, without integrating into the genome of the host cell.

Generally speaking, adenoviral DNA is typically very stable and remainsepisomal (e.g., extrachromosomal), unless transformation ortumorigenesis has occurred. In addition, adenoviral vectors can bepropagated to high yields in well-defined production systems which arereadily amenable to pharmaceutical scale production of clinical gradecompositions. These characteristics and their well-characterizedmolecular genetics make recombinant adenoviral vectors good candidatesfor use as vaccine carriers. Typically, the production of recombinantadenoviral vectors relies on the use of a packaging cell line which iscapable of complementing the functions of adenoviral gene products thathave been either deleted or engineered to be nonfunctional.

Presently, two well-characterized human subgroup C adenovirus serotypes(i.e., hAd2 and hAd5) are widely used as the sources of the viralbackbone for most of the adenoviral vectors that are used for genetherapy. Replication-defective human adenoviral vectors have also beentested as vaccine carriers for the delivery of a variety of immunogensderived from a variety of infectious agents (e.g., viruses, parasites,or bacterial pathogens) and tumor cells, including tumor-associatedantigens (TAAs). Studies conducted in experimental animals (e.g.,rodents, canines and nonhuman primates) indicate that recombinantreplication-defective human adenoviral vectors carrying transgenesencoding immunogens derived from the E6 and E7 oncoproteins of humanpapillomavirus (HPV-16) (He, Z et al., (2001) Virology, 270:3583-3590,the rabies virus glycoprotein (Xiang, Z. et al (1996) Virology,219:220-227), the circumsporozoite protein of Plasmodium falciparumRodriguez, E. et al. (1997) J. Immunol. 158:1268-1274) as well as otherheterologous antigens elicit both humoral and cell-mediated immuneresponses against the transgene product. Generally speaking,investigators have reported success using human adenoviral vectors asvaccine carriers in nonhuman experimental systems by either using animmunization protocols that utilizes high doses of recombinantadenoviral vectors that are predicted to elicit immune responses; or byusing immunization protocols which employ the sequential administrationof adenoviral vectors that are derived from different serotypes butwhich carry the same transgene product as boosting immunizations(Mastrangeli, et al., Human Gene Therapy, 7: 79-87 (1996).

However, it is predicted that vaccine carriers derived from ubiquitoushuman serotypes, such as types 2 and 5, will encounter preexistinghumoral and cellular immunity in the human population. Thus, althoughreplication-defective recombinant human adenoviruses have beensuccessfully employed as vaccine carriers in experimental systemsemploying rodent, canine, and nonhuman primate hosts; human innate andadaptive immunity is expected to significantly limit the utility ofthese serotypes as vaccine carriers. This expectation is based on thefact that subgroup C, which includes type 2 and type 5, adenoviralinfection is endemic in the human population. As a consequence, themajority of humans seroconvert within the first five years of life asthe result of a natural infection. Thus, vectors derived from virusesthat naturally infect and replicate in humans may not be optimalcandidates for use as vaccine carriers.

Another problem associated with the use of human adenoviral-derivedvectors is the risk that the production method used to propagate therecombinant viruses will give rise to vector stocks that arecontaminated with replication competent adenovirus (RCA). This is causedby homologous recombination between overlapping sequences from therecombinant vector and the adenoviral genes that are present in theE1-complementing helper cell lines such as human 293 (Graham, F. L. etal, (1977) J. Gen. Virol. 36:59-72.) cells. The presence of RCA invector stocks prepared for use in clinical trials constitutes a safetyrisk because it can promote the mobilization and spread of thereplication defective virus. Spread of the defective virus can aggravatethe host immune response and cause other adverse immunopathologicalconsequences (Fallux, F. J., et al. Human Gene Therapy 9: 1909-1917(1998). Accordingly, the Food and Drug Administration (FDA) and otherregulatory bodies have promulgated guidelines which establish limits onthe levels of RCA that can be present in vector preparations intendedfor clinical use. The intent of imposing RCA limits is to ensure limitedexposure of patients to replicating adenovirus in compositions that areused in clinical trials.

Thus, there continues to be a need for the development of adenoviralvaccine carriers that are suitable for use in mammalian hosts which are:easy to manipulate, amenable to pharmaceutical scale production and longterm storage, capable of high-level replication in human complementationcell lines, highly immunogenic, devoid of neutralizing B cell epitopesthat cross-react with the common serotypes of human adenoviruses, complywith the safety RCA standards promulgated by regulatory agencies, andwhich are amenable for use in prime/boost protocols that are suitablefor use in humans.

SUMMARY OF THE INVENTION

The present invention relates to recombinant replication-defectiveadenovirus vectors derived from chimpanzee adenoviruses and methods forgenerating chimpanzee adenoviral vectors in human E1-expressing celllines. The invention also provides methods for generating clinical gradevector stocks suitable for use in humans and means for using thedisclosed vectors as vaccine carriers to elicit protective and/ortherapeutic immune responses. The invention further provides methods forusing the recombinant adenoviruses of the invention to prepare vaccinecompositions designed to delivery, and direct the expression of,transgenes encoding immunogens. In one embodiment, the inventioncontemplates the use of the disclosed vectors as vaccine carriers forthe administration of vaccines comprising transgenes encoding immunogensderived from an infectious agent. In a second embodiment, the inventioncontemplates the use of the disclosed vectors to prepare and administercancer vaccines. In a particular embodiment, the invention contemplatesthe preparation and administration of a cancer vaccine comprising atransgene encoding a TAA.

In one aspect, the invention discloses the complete genomic sequence offive chimpanzee adenoviruses (ChAds), referred to herein as ChAd3 (SEQID NO: 1) (FIGS. 5A-5K), ChAd6 (SEQ ID NO: 2) (FIGS. 6A-6K, CV32 (SEQ IDNO: 3) (FIGS. 7A-7K), CV33 (SEQ ID NO: 4) (FIGS. 8A-8K), and CV23 (SEQID NO: 5) (FIGS. 9A-9J).

ChAd3 and ChAd6 represent novel adenoviruses isolated according to themethods disclosed herein. The genomes of the ChAd3 and ChAd6 are 37741and 36648 base pairs in length, respectively. The ChAd3 hexon gene (SEQID NO: 41) comprises nucleotides (nt) 19086-21965 of SEQ ID NO: 1(exclusive of stop codon) and the ChAd3 fiber gene (SEQ ID NO: 42)comprises nt 32805-34487 of SEQ ID NO: 1 (exclusive of stop codon). TheChAd6 hexon gene comprises nt 18266-21124 (SEQ ID NO: 43) of SEQ ID NO:2 (exclusive of stop codon) and its fiber gene (SEQ ID NO: 44) comprisesnt 32218-33552 of SEQ ID NO:2 (exclusive of stop codon). Based onsequence homology deduced from a multiple sequence alignment offull-length hexon peptides, ChAd3 has been classified into humansubgroup C and ChAd6 has been classified into human subgroup E.

The genomes of the CV32, CV33 and CV23 adenoviruses are 36,606, 36,535,and 32,020 base pairs in length, respectively. CV32 (Pan 6) (ATCC®(American Type Culture Collection) N. VR-592), CV33 (Pan 7) (ATCC®(American Type Culture Collection) N. VR-593) and CV23 (Pan 5) (EsoterixInc.,) have all been determined to be related to human Ad4 (hAd4)(subgroup E) (Wigand, R et al. Intervirology 1989, 30:1-9). However,based on hexon sequence alignment CV32 has subsequently characterized asbeing more closely analogous to human subgroup D members than to hAd4.

In a second aspect, the invention provides nucleotide sequences for thefiber and hexon genes of 21 additional chimpanzee adenoviruses (ChAd20,ChAd4, ChAd5, ChAd7, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19,ChAd8, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44,ChAd63 and ChAd82) isolated according to the methods disclosed herein.

The fiber gene nucleotide sequences for ChAd20, ChAd4, ChAd5, ChAd7,ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, are set forth in FIGS.10-19, respectively, and are referred to herein as SEQ ID NOS: 6-15:(SEQ ID NO: 6, ChAd20); (SEQ ID NO: 7, ChAd4); (SEQ ID NO: 8, ChAd5);(SEQ ID NO: 9, ChAd7); (SEQ ID NO: 10, ChAd9); (SEQ ID NO: 11, ChAd10);(SEQ ID NO: 12, ChAd11); (SEQ ID NO: 13, ChAd16) (SEQ ID NO: 14, ChAd17)and (SEQ ID NO: 15, ChAd19).

The fiber gene nucleotide sequences for ChAd8, ChAd22, ChAd24, ChAd26,ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 referred toherein as: (SEQ ID NO: 58, ChAd8), (SEQ ID NO: 60, ChAd22), (SEQ ID NO:62, ChAd24), (SEQ ID NO: 64, ChAd26), (SEQ ID NO: 66, ChAd30), (SEQ IDNO: 68, ChAd31), (SEQ ID NO: 70, ChAd37), (SEQ ID NO: 72, ChAd38), (SEQID NO: 74, ChAd44), (SEQ ID NO: 76, ChAd63) and (SEQ ID NO: 78, ChAd82)and are set forth in the sequence listing.

The hexon gene nucleotide sequences for ChAd20, ChAd4, ChAd5, ChAd7,ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, are set forth in FIGS.21-30, respectively, and are referred to herein as SEQ ID NOS: 16-25:(SEQ ID NO: 16, ChAd20); (SEQ ID NO: 17, ChAd4); (SEQ ID NO: 18, ChAd5);(SEQ ID NO: 19, ChAd7); (SEQ ID NO: 20, ChAd9); (SEQ ID NO: 21, ChAd10);(SEQ ID NO: 22, ChAd11); (SEQ ID NO: 23, ChAd16); (SEQ ID NO: 24,ChAd17) and (SEQ ID NO: 25, ChAd19).

The hexon gene nucleotide sequences for ChAd8, ChAd22, ChAd24, ChAd26,ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 referred toherein as: (SEQ ID NO: 97, ChAd8), (SEQ ID NO: 99, ChAd22), (SEQ ID NO:101, ChAd24), (SEQ ID NO: 103, ChAd26), (SEQ ID NO: 105, ChAd30), (SEQID NO: 107, ChAd31), (SEQ ID NO: 109, ChAd37), (SEQ ID NO: 111, ChAd38),(SEQ ID NO: 113, ChAd44), (SEQ ID NO: 115, ChAd63) and (SEQ ID NO: 117,ChAd82) and are set forth in the sequence listing.

In a third aspect, the invention provides amino acid sequences for thefiber and hexon proteins of 21 additional chimpanzee adenoviruses(ChAd20, ChAd4, ChAd5, ChAd7, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17,ChAd19, ChAd8, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38,ChAd44, ChAd63 and ChAd82) isolated according to the methods disclosedherein.

The fiber proteins which are disclosed and claimed here as are referredto as: (SEQ ID NO: 83, ChAd3), (SEQ ID NO: 84, ChAd6), (SEQ ID NO: 48,ChAd20), (SEQ ID NO: 49, ChAd4), (SEQ ID NO: 50, ChAd5), (SEQ ID NO: 51,ChAd7), (SEQ ID NO: 52, ChAd9), (SEQ ID NO: 53, ChAd10), (SEQ ID NO: 54,ChAd11), (SEQ ID NO: 55, ChAd16), (SEQ ID NO: 56, ChAd17), (SEQ ID NO:57, ChAd19), (SEQ ID NO: 59, ChAd8), (SEQ ID NO: 61, ChAd22), (SEQ IDNO: 63, ChAd24), (SEQ ID NO: 65, ChAd26), (SEQ ID NO: 67, ChAd30), (SEQID NO: 69, ChAd31), (SEQ ID NO: 71, ChAd37), (SEQ ID NO: 73, ChAd38),(SEQ ID NO: 75, ChAd44), (SEQ ID NO: 77, ChAd63) and (SEQ ID NO: 79,ChAd82). FIGS. 20A-20G provides an alignment comparing the amino acidsequences of the fiber proteins disclosed and claimed herein with theamino acid sequences of the fiber proteins of: C1 (SEQ ID NO: 85), CV68(SEQ ID NO: 86), Pan5 (alternatively referred to as CV23) (SEQ ID NO:80), Pan6 (alternatively referred to as CV32) (SEQ ID NO: 81), and Pan7(alternatively referred to as CV33) (SEQ ID NO: 82).

The hexon proteins which are disclosed and claimed here as are referredto as: (SEQ ID NO: 122, ChAd3), (SEQ ID NO: 123, ChAd6), (SEQ ID NO: 87,ChAd20), (SEQ ID NO: 88, ChAd4), (SEQ ID NO: 89, ChAd5), (SEQ ID NO: 90,ChAd7), (SEQ ID NO: 91, ChAd9), (SEQ ID NO: 92, ChAd10), (SEQ ID NO: 93,ChAd11), (SEQ ID NO: 94, ChAd16), (SEQ ID NO: 95, ChAd17), (SEQ ID NO:96, ChAd19), (SEQ ID NO: 98, ChAd8), (SEQ ID NO: 100, ChAd22), (SEQ IDNO: 102, ChAd24), (SEQ ID NO: 104, ChAd26), (SEQ ID NO: 106, ChAd30),(SEQ ID NO: 108, ChAd31), (SEQ ID NO: 110, ChAd37), (SEQ ID NO: 112,ChAd38), (SEQ ID NO: 114, ChAd44), (SEQ ID NO: 116, ChAd63) and (SEQ IDNO: 118, ChAd82). FIGS. 31A-31J provide a comparison of the amino acidsequences of the hexon proteins disclosed and claimed herein with theamino acid sequences of the hexon proteins of: C1 (SEQ ID NO: 124), CV68(SEQ ID NO: 125), Pan5 (alternatively referred to as CV23) (SEQ ID NO:119), Pan6 (alternatively referred to as CV32) (SEQ ID NO: 120), andPan7 (alternatively referred to as CV33) (SEQ ID NO: 121). A multiplesequence alignment of hexon proteins allows an artisan to perform aphylogenetic analysis of that is consistent with the proposedclassification of human adenoviral serotypes (Rux, J. J., et al (2003)J. Virol. 77:9553-9566).

In an alternative aspect, the invention further provides 21 additionalchimpanzee adenovirus isolates. Samples comprising ChAd20, ChAd4, ChAd5,ChAd7, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17 and ChAd19 were depositedon Dec. 12, 2003 with the European Collection of Cell Cultures (ECACC®,Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom) as anoriginal deposit under the Budapest Treaty. The deposits were assignedaccession numbers: 03121201 (ChAd4), 03121202 (ChAd5), 03121203 (ChAd7),03121204 (ChAd9), 03121205 (ChAd10), 03121206 (ChAd11), 03121207(ChAd16), 03121208 (ChAd17), 03121209 (ChAd19) and 03121210 (ChAd20).

Samples comprising ChAd8, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31,ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 were deposited with the ECACC®(European Collection of Cell Cultures, Porton Down, Salisbury,Wiltshire, SP4 0JG, United Kingdom) as an original deposit under theBudapest Treaty on Jan. 12, 2005. These deposits were assigned accessionnumbers: 05011201 (ChAd8), 05011202 (ChAd22), 05011203 (ChAd24),05011204 (ChAd26), 05011205 (ChAd30), 05011206 (ChAd31), 05011207(ChAd37), 05011208 (ChAd38), 05011209 (ChAd44), 05011210 (ChAd63) and05011211 (ChAd82).

These deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. §112. All restrictions on theavailability to the public of the deposited material will be irrevocablyremoved, except for the requirements specified in 37 C.F.R. §1.808(b),upon the granting of a patent.

In an additional aspect, the invention also providesreplication-defective recombinant adenoviral vectors which are capableof infecting mammalian cells, preferably human cells, and directingexpression of encoded transgene product(s). As demonstrated herein, thedisclosed vectors are suitable for use as vaccine carriers for thedelivery of transgenes comprising immunogens against which an immuneresponse is desired. In particular embodiments, the invention providesrecombinant replication-defective chimpanzee adenoviral vectors that arecapable of high-level replication in human E1-expressing (i.e.,packaging) cell lines. In one embodiment, the invention providesrecombinant adenoviruses that are capable of replicating in PER.C6™cells.

Generally speaking, the recombinant vectors encompassed by the inventionprovide vaccine carriers that will evade pre-existing immunity to theadenovirus serotypes that are typically encountered in the humanpopulation. More specifically, the recombinant vectors of the inventioncomprise vector backbone sequences which are shown herein to be devoidof neutralizing B epitopes that cross-react with the common serotypes ofhuman adenoviral derived vectors.

The invention further provides group-specific shuttle vectors thatinclude an adenoviral portion and a plasmid portion, wherein saidadenoviral portion generally comprises: a) viral left end (ITR andpackaging signal), part of the pIX gene and viral genome right end; andb) a gene expression cassette. The group-specific shuttle vectors aredesigned to exploit the nucleotide sequence homology which is observedbetween adenoviruses that are assigned to the same serotype subgroup(i.e., subgroups A, B, C, D or E), and can be used to manipulate thenucleotide sequences disclosed herein and/or to clone other chimpanzeeadenoviruses belonging to the same subgroup generating an adenoviruspre-plasmid containing a chimp adenoviral genome deleted of E1 region.

Other aspects of this invention include host cells comprising theadenoviral vaccine vectors and/or the adenovirus pre-plasmid vectors,methods of producing the vectors comprising introducing the adenoviralvaccine vector into a host cell which expresses adenoviral E1 protein,and harvesting the resultant adenoviral vaccine vectors. In a particularembodiment, the invention provides a method of producing areplication-defective chimpanzee adenoviral vector comprisingintroducing one of the disclosed adenoviral vectors into an adenoviralE-1 expressing human cell, and harvesting the resulting recombinantadenoviruses.

Another aspect of the invention also provides vaccine compositions whichcomprise an adenoviral vector of the invention. Compositions comprisingrecombinant chimpanzee adenoviral vectors may be administered alone orin combination with other viral- or non-viral-based DNA/proteinvaccines. They also may be administered as part of a broader treatmentregimen. These compositions can be administered to mammalian hosts,preferably human hosts, in either a prophylactic or therapeutic setting.As shown herein, administration of the disclosed vaccine compositions,either alone or in a combined modality, such as a prime boost regimen ormultiple injections of serologically distinct Ad vectors results in theinduction of an immune response in a mammal that is capable ofspecifically recognizing the immunogen encoded by the transgene.

One of the methods disclosed and claimed herein, comprises administeringto a mammal (that is either naïve or primed to be immunoreactive to atarget antigen), a sufficient amount of a recombinant chimpanzeeadenoviral vector, containing at least a functional deletion of itswild-type E1 gene, carrying a sequence comprising a promoter capable ofdirecting expression of a nucleotide sequence encoding the least onetarget antigen, wherein administration of the recombinant vector elicits(or primes) an antigen-specific immune response.

In one embodiment, the invention provides a method designed to induce animmune response (prophylactic or therapeutic) against an infectiousagent (e.g., a viral or bacterial pathogen or a mammalian parasite). Ina second embodiment, the invention provides a method designed to inducean immune response in a mammal that will break tolerance to aself-antigen, such as a TAA. This aspect of the invention contemplatesthe use of the disclosed vectors as a vaccine carrier for thepreparation and administration of cancer vaccines.

Yet other embodiments and advantages of the present invention will bereadily apparent from the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing which summarizes the cloning strategy usedto construct a ChAd6 shuttle vector (pARS ChAd6-3).

FIG. 2 is a schematic drawing which illustrates the cloning strategyused to clone the ChAd6 viral genome by homologous recombination in E.coli strain BJ5183.

FIG. 3 is a schematic drawing illustrating the elements of various ChAd6shuttle plasmids including: pARS ChAd6-3 GAG; pARS ChAd6-3 SEAP; pARSChAd6-3 EGFP; and pARS ChAd6-3 NS MUT.

FIG. 4 is a schematic drawing which illustrates the homologousrecombination scheme utilized to clone the ChAd6 ΔE1 expression vectors.

FIGS. 5A-5K provides the genomic nucleotide sequence of ChAd3 (SEQ IDNO: 1).

FIGS. 6A-6K provides the genomic nucleotide sequence of ChAd6 (SEQ IDNO: 2).

FIGS. 7A-7K provides the genomic nucleotides sequence of CV32 (SEQ IDNO: 3).

FIGS. 8A-8K provides the genomic nucleotide sequence of CV33 (SEQ ID NO:4).

FIGS. 9A-9J provides the genomic nucleotide sequence of CV23 (SEQ ID NO:5).

FIG. 10 provides the nucleotide sequence of the fiber gene of ChAd20(SEQ ID NO: 6).

FIG. 11 provides the nucleotide sequence of the fiber gene of ChAd4 (SEQID NO: 7).

FIG. 12 provides the nucleotide sequence of the fiber gene of ChAd5 (SEQID NO: 8).

FIG. 13 provides the nucleotide sequence of the fiber gene of ChAd7 (SEQID NO: 9).

FIG. 14 provides the nucleotide sequence of the fiber gene of ChAd9 (SEQID NO: 10).

FIG. 15 provides the nucleotide sequence of the fiber gene of ChAd10(SEQ ID NO: 11).

FIG. 16 provides the nucleotide sequence of the fiber gene of ChAd11(SEQ ID NO: 12).

FIG. 17 provides the nucleotide sequence of the fiber gene of ChAd16(SEQ ID NO: 13).

FIG. 18 provides the nucleotide sequence of the fiber gene of ChAd17(SEQ ID NO: 14).

FIG. 19 provides the nucleotide sequence of the fiber gene of ChAd19(SEQ ID NO: 15).

FIGS. 20A-20G provides a comparison of the amino acid sequences of thefiber proteins of: ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9,ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26,ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 with thereference fiber protein sequences from C1 (SEQ ID NO: 85), CV68 (SEQ IDNO: 86), PANS (also referred to as CV23) (SEQ ID NO: 80), PAN6 (alsoreferred to as CV32) (SEQ ID NO: 81) and Pan7 (also referred to as CV33)(SEQ ID NO: 82).

FIG. 21 provides the nucleotide sequence of the hexon gene of ChAd20(SEQ ID NO: 16).

FIG. 22 provides the nucleotide sequence of the hexon gene of ChAd4 (SEQID NO: 17).

FIG. 23 provides the nucleotide sequence of the hexon gene of ChAd5 (SEQID NO: 18).

FIG. 24 provides the nucleotide sequence of the hexon gene of ChAd7 (SEQID NO: 19).

FIG. 25 provides the nucleotide sequence of the hexon gene of ChAd9 (SEQID NO: 20).

FIG. 26 provides the nucleotide sequence of the hexon gene of ChAd10(SEQ ID NO: 21).

FIG. 27 provides the nucleotide sequence of the hexon gene of ChAd11(SEQ ID NO: 22).

FIG. 28 provides the nucleotide sequence of the hexon gene of ChAd16(SEQ ID NO: 23).

FIG. 29 provides the nucleotide sequence of the hexon gene of ChAd17(SEQ ID NO: 24).

FIG. 30 provides the nucleotide sequence of the hexon gene of ChAd19(SEQ ID NO: 25).

FIGS. 31A-31J provides a comparison of the amino acid sequences of thehexon proteins of ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9,ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26,ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 with thereference fiber protein sequences from C1 (SEQ ID NO: 124), CV68 (SEQ IDNO: 125), PANS (also referred to as CV23) (SEQ ID NO: 119), PAN6 (alsoreferred to as CV32) (SEQ ID NO: 120) and Pan7 (also referred to asCV33) (SEQ ID NO: 121).

FIG. 32 provides a listing of the artificial sequences SEQ ID NOS: 26-40and SEQ ID NOS: 45 and 46, including oligomers and primers, disclosedherein.

FIG. 33 is a graphic representation of the immunization break-point ofChAd vectors belonging to different serotype subgroups (i.e., subgroupsC, E and D). The lowest dose eliciting a measurable immune response wasdetermined by performing titration experiments in mice immunized withgag-expressing ChAd3, ChAd11, ChAd20, CV33, CV68, ChAd6, ChAd9, ChAd10,CV32, ChAd4, ChAd7 and ChAd16 vectors.

FIG. 34 provides a graphic representation of a CEA-specific T cellresponse elicited in rhesus macaques immunized sequentially with a humanadenoviral vector (MRKAd5 RhCEA) followed by a chimpanzee adenoviralvector (CV33 RhCEA) after 12 week interval. The immune responses wereevaluated by IFN-γ ELISPOT assay, and the data illustrate the number ofspot-forming cells (SFC) per million peripheral blood mononuclear cells(PBMC) following incubation in the absence (DMSO) and presence of rhesusCEA C and D peptide pools.

FIG. 35 provides a phylogenetic tree of human and chimpanzeeadenoviruses of deduced from a multiple sequence alignment offull-length hexon peptide sequences using PAUPSEARCH (Wisconsin PackageVersion 10.3, Accelrys Inc.) and visualized and manipulated withTREEVIEW.

FIG. 36 is a graphic representation of immunization results obtained inresponse to the administration of ChAd3 and hAd5 gag vectors to micewhich were pre-exposed to hAd5. Cell-mediated immunity was evaluated 3weeks post-immunization by IFN-γ ELISPOT using purified splenocytes.

FIG. 37 is a graphic representation of kinetics of anti-CEA CMI elicitedin human CEA transgenic mice immunized with ChAd3hCEA and Ad5hCEA. CMIwas evaluated by ICS of PBMC stimulated with CEA peptide pool. Theresults are expressed as % of IFNγ⁺ CD8⁺/total PBMC.

FIG. 38 A-D is a graphic representation of the efficiency of infectionof different human primary cells exposed to moi 50, 250 and 1250 ofdifferent ChAd vectors expressing EGFP and belonging to differentsubgroups (B, C, D, E). The results are expressed as % of fluorescentcells/on total cells.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the specification and appended claims, the followingdefinitions and abbreviations apply:

The term “cassette” refers to a nucleic acid molecule which comprises atleast one nucleic acid sequence that is to be expressed, along with itstranscription and translational control sequences. Changing thecassette, will cause the vector into which is incorporated to direct theexpression of different sequence or combination of sequences. In thecontext of the present invention, the nucleic acid sequences present inthe cassette will usually encode an immunogen. Because of therestriction sites engineered to be present at the 5′ and 3′ ends, thecassette can be easily inserted, removed or replaced with anothercassette.

The term “cis-acting element” refers to nucleotide sequences whichregulate genes to which they are attached. Cis-acting elements presentin DNA regulate transcription, and those transcribed into mRNA canregulate RNA processing, turnover and protein synthesis.

The term “vector” refers to some means by which DNA fragments can beintroduced into a host organism or host tissue. There are various typesof vectors including plasmid, virus (including adenovirus),bacteriophages and cosmids.

The term “promoter” refers to a recognition site on a DNA strand towhich an RNA polymerase binds. The promoter forms an initiation complexwith RNA polymerase to initiate and drive transcriptional activity. Thecomplex can be modified by activating sequences such as enhancers, orinhibiting sequences such as silencers.

The term “pharmaceutically effective amount” refers to an amount ofrecombinant adenovirus that is effective in a particular route ofadministration to transduce host cells and provide sufficient levels oftransgene expression to elicit an immune response.

The term “replication-competent” recombinant adenovirus (AdV) refers toan adenovirus with intact or functional essential early genes (i.e.,E1A, E1B, E2A, E2B and E4). Wild type adenoviruses are replicationcompetent.

The term “replication-defective” recombinant AdV refers to an adenovirusthat has been rendered to be incapable of replication because it hasbeen engineered to have at least a functional deletion, or a completeremoval of, a gene product that is essential for viral replication. Therecombinant chimpanzee adenoviral vectors of the invention arereplication-defective.

The term “mammalian” refers to any mammal, including a human being.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length of the genome(e.g., about 36 kbp), the full-length of an open reading frame of agene, protein, subunit, or enzyme [see, e.g., the tables providing theadenoviral coding regions], or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Similarly, “percent sequenceidentity” may be readily determined for amino acid sequences, over thefull-length of a protein, or a fragment thereof. Suitably, a fragment isat least about 8 amino acids in length, and may be up to about 700 aminoacids. Examples of suitable fragments are described herein.

Identity is readily determined using such algorithms and computerprograms as are defined herein at default settings. Preferably, suchidentity is over the full length of the protein, enzyme, subunit, orover a fragment of at least about 8 amino acids in length. However,identity may be based upon shorter regions, where suited to the use towhich the identical gene product is being put.

In general, adenoviral constructs, gene constructs are named byreference to the genes contained therein. For example, “pChAd3 ΔE1gag”refers to a plasmid construct which comprises a ChAd3 chimpanzeeadenoviral genome deleted of the E1 region. In this plasmid, the E1region is replaced by an immunogen expression cassette comprising an HIVgag gene under the control of a human CMV promoter followed by a bovinegrowth hormone polyadenylation signal. Similarly, pCV33DE1-E3 NSmut,refers to a second plasmid construct disclosed herein which comprises aCV33 chimpanzee adenoviral genome, deleted of the E1 and E3 regions,which is replaced by an immunogen expression cassette comprising HCVnon-structural genes under the control a human CMV promoter followed bya bovine growth hormone polyadenylation signal.

The abbreviation “Ag” refers to an antigen.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

Adenoviruses (Ads) are noneveloped, icosahedral viruses that have beenidentified in several avian and mammalian hosts. Human Ads (hAd) belongto the Mastadenovirus genus which includes all known human and many Adsof animal (e.g., bovine, porcine, canine, murine, equine, simian andovine) origin. Human adenoviruses are divided into six subgroups (A-F)based on a number of biological, chemical, immunological and structuralcriteria which include hemagglutination properties of rat and rhesusmonkey erythrocytes, DNA homology, restriction enzyme cleavage patterns,percentage G+C content and oncogenicity (Straus, 1984, In TheAdenoviruses, ed. H. Ginsberg, pps. 451-498, New York: Plenus Press, andHorwitz, 1990 In Virology, eds. B. N. Fields and D. M. Knipe, pps.1679-1721). To date, 51 distinct serotypes have been recognized andgrouped into subgroups on the basis of their hemagglutination propertiesand biophysical and biochemical criteria.

The adenoviral virion has an icosahedral symmetry and, depending on theserotype, a diameter of 60-90 nm. The icosahedral capsid consists threemajor proteins, hexon (II), penton base (III) and a knobbed fiber (IV)as well as a number of minor proteins (i.e., VI, VIII, IX, Ma and IVa2)(W. C. Russel, J. Gen. Virol., 81: 2573-2604 (2000). One aspect of thepreexisting immunity that is observed in humans is humoral immunity,which can result in the production and persistence of antibodies thatare specific for viral proteins. The humoral response elicited byadenovirus is mainly directed against the major structural proteins:hexon, penton and fiber.

Published reports have established that titers comprising antibodiesagainst multiple serotypes are common (Dambrosio, E. (1982) J. Hyg.(London) 89: 209-219) and that a substantial portion of the preexistingtiters have neutralizing activity. Neutralizing immunity to adenovirusis type specific, and infection with a particular serotype of adenovirusconfers immunity only to that serotype. Several reports have suggestedthat antibodies directed towards the hexon are the strongest and themost neutralizing (Toogood, C. I. A., Crompton, J. and Hay R. T. (1992)J. Gen. Virol. 73, 1429-1435). Therefore, it is reasonable to assumethat the epitopes responsible for type-specific neutralization arelocated within seven hypervariable regions identified by alignment ofthe hexon sequences deriving from different serotypes. (Crawford-Miksza,L and D. P. Schnurr. (1996) J. Virol. 70:1836-1844).

A direct correlation between the presence of type-specific neutralizingantibodies and the inability to elicit an immune response with a vectorbased on the same serotype has been established by different methodsincluding the passive transfer of immune sera from treated to naïveanimals. Generally speaking, preexisting humoral immunity for a specificviral serotype reduces the therapeutic efficacy of the vectoradministration. Moreover, the administration of a vector based on aspecific viral serotype elicits an immune-response against the vectorthat prevents the re-administration of the same serotype.

In a particular embodiment, the invention provides a method ofcircumventing the adverse effects associated with the consequences ofpreexisting immunity to common serotypes of hAds. More specifically, theinvention contemplates the use of chimpanzee adenoviral vectorscharacterized by a serotype that does not circulate in humans.Accordingly, the invention provides adenoviral (Chad) vectors which lackneutralizing B-cell epitopes that cross react with those of common humanserotypes as a vaccine carrier.

Although it has been reported that adenoviral-specific cell mediatedimmunity (CMI) can be cross-reactive, vaccination studies based onrepeated injections of multiple serotypes demonstrated a higherefficiency than immunization schedules based on a single vector. Theseexperiments further demonstrate that the main limitation of a vectoradministration for vaccine purposes is the humoral pre-existing immunityagainst the vector. Potential solutions to the problems associated withthe use of a human adenovirus as a vaccine carrier include theadministration of a higher dose of an adenovirus (e.g., a subgroup Cserotype) that is predicted to encounter a preexisting immune response,and the use of vectors based on rare human serotypes. However, the useof higher doses of vaccine increases the cost of the vaccine and risk ofundesirable side effects and the results of preclinical testing suggestthat human alternate serotypes are less immunogenic than hAd5 and hAd6.

In an attempt to avoid the problems of host humoral and cellular immuneresponses against the adenoviral backbone elements of the vector, and tominimize the risk of using human adenovirus-derived vector stocks thatmay be contaminated with replication-competent adenoviruses (RCA),several nonhuman adenoviruses have been characterized and developed asvaccine carriers (Soudois, C. et al (2000) J. Virology, 74:10639-10649;Farina, S. F. et al (2001) J. Virology, 75:11603-11613; Cohen, C. J. etal (2002) J. Gen. Virology, 83:151-155.) The premise underlying the useof nonhuman adenoviral sequences to circumvent the problems associatedwith preexisting immunity is based on the observation that neutralizingantibodies to common human adenovirus serotypes are unlikely tocross-neutralize nonhuman viruses. However, the incompatibility of viraland cellular factors imposes a practical limitation on the vast majorityof alternative vector systems (bovine, ovine, canine) which arecharacterized by the disadvantage of having to be propagated innon-human cell lines.

Wilson et al. have published a report describing the characterization ofa replication-defective vector based on chimpanzee adenovirus type 68(CV68) C68, which was originally isolated from a mesenteric lymph nodeof a chimpanzee (Basnight, M., et. al. (1971) Am. J. Epidemiol.94:166-171.), CV68 was fully sequenced and found to be similar inoverall structure to human adenoviruses (Farina, S. F. et al., J. Virol.75(23): 11603-11613 (2001). The genome of the virus is 36,521 base pairsin length and has been described as being most similar to subgroup E ofhuman adenoviruses, with 90% identity to most human Ad4 open readingframes that have been sequenced. The CV68 ITRs are 130 base pairs inlength, and all of the major adenoviral early and late genes arepresent. CV68 is characterized by a serotype that does not circulate inhumans and which lacks neutralizing B cell epitopes that cross-reactwith those of common human serotypes. Although Chimpanzee adenonvirusesare similar to human adenoviruses cross-reactive neutralizing immunityagainst chimpanzee serotypes has not been documented in humans (Farina,S. F. et al. J. Virol. (2001) 75(23):11603-13).

The recombinant vectors derived from CV68 are described as beingsufficiently similar to human serotypes to support transduction of cellsexpressing the coxsackievirus and adenovirus receptor (Cohen, C. et al.,J. Gen. Virol. 83: 151-155 (2002). Significantly, CV68 is characterizedby a sufficient level of similarity to human adenoviruses to support itsreplication 293 cells which harbor E1 from human adenovirus type 5(Farina, S. F. et al., J. Virol. 75(23): 11603-11613 (2001).Furthermore, based on the observation that the flanking sequences of thehuman serotype 5 E1 are nonhomologous with those of the CV68-derivedvector sequences, it is predicted that homologous recombination will notoccur. Thus, it has been predicted that there is a low likelihood thatCV68-derived vaccine stocks will be contaminated with RCA.

The same group of investigators subsequently reported the use ofCV68-derived adenoviral sequences as a vaccine carrier for induction ofantibodies to the rabies virus glycoprotein in mice. Areplication-defective version of CV68 was created by replacing the E1Aand E1B genes with a minigene cassette. Mice immunized with anE1-deletion-containing adenoviral recombinant (AdC68rab.gp) comprising atransgene product encoding the rabies virus glycoprotein developedprotective immunity to rabies virus and remained resistant to challengewith an otherwise lethal dose of rabies virus (Xiang, Z et al., J.Virol. 76(5): 2667-2675 (2002). A second CV68 construct expressing acodon-optimized, truncated form of gag of HIV-1 was recently reported toinduce a vigorous gag-specific CD8⁺ T cell response in mice. Thevaccine-induced response was shown to provide protection to challengewith a vaccinia gag recombinant virus (Fitzgerald, J. C. et al., J.Immunol. 170: 1416-1422 (2003). Experimental vaccination of micepreimmunized to human adenovirus serotype 5 with CV68gag or Ad5gagvectors demonstrated a more pronounced reduction of gag-specific T cellsand protection against viral challenge elicited by Ad5 than by CV68vaccine. The reduction in efficacy of C68gag vaccine was attributed to across-reactivity of Ad5-specific CD8+ T cells (Id.).

Considered together this data suggests that simian-derivedreplication-defective adenoviral vectors may be more suitable for use ashuman vaccine carriers than vectors based on common human serotypes. Asshown herein, the results of experiments in which mice that werestrongly immunized against human Ad5 (FIG. 36) can be immunized withChAd3-gag adenoviral vectors indicate the preexisting anti-human Ad5immunity did not reduce the gag-specific CMI response elicited by theChAd vectors. These results are consistent with the conclusion thathuman Ad5 cross-reactive B and T-cell epitopes are not present in ChAd3-or ChAd6 vectors.

Generally speaking, the adenoviral genome is very well characterized anddespite the existence of several distinct serotypes, there is somegeneral conservation in the overall organization of the adenoviralgenome with specific functions being similarly positioned. Thenucleotide sequences of the chimpanzee adenoviruses C1 and CV68disclosed by Wilson et al., and the location of the E1A, E1B, E2A, E2B,E3, E4, L1, L2, L3, L4 and L5 genes of each virus are provided in U.S.Pat. No. 6,083,716 (Chimpanzee Adenovirus Vectors), and PCT publishedapplication WO 03/000851 (Methods for Rapid Screening of BacterialTransformants and Novel Simion Adenoviral Proteins), the teachings ofwhich are incorporated herein by reference.

Each extremity of the adenoviral genome comprises a sequence known as aninverted terminal repeat (ITRs), which is necessary for viralreplication. The virus also comprises a virus-encoded protease, which isnecessary for processing some of the structural proteins required toproduce infectious virions. The structure of the adenoviral genome isdescribed on the basis of the order in which the viral genes areexpressed following host cell transduction. More specifically, the viralgenes are referred to as early (E) or late (L) genes according towhether transcription occurs prior to or after onset of DNA replication.In the early phase of transduction, the E1, E2, E3 and E4 genes ofadenovirus are expressed to prepare the host cell for viral replication.The virus can be rendered replication defective by deletion of theessential early-region 1(E1) of the viral genome. Brody et al, 1994 AnnN Y Acad Sci., 716:90-101. During the late phase, expression of the lategenes L1-L5, which encode the structural components of the virusparticles is switched on. All of the late genes are under the control ofa single promoter and encode proteins including the penton (L2), thehexon (L3), the 100 kDa scaffolding protein (L4), and the fiber protein(L5), which form the new virus particle into which the adenoviral DNAbecomes encapsidated. Depending on the serotype of the virus,10,000-100,000 progeny adenovirus particles can be generated in a singlehost cell. Ultimately, the adenoviral replication process causes lysisof the cells.

The replication-defective adenoviral vectors disclosed herein wereconstructed by deletion of specific nucleotide sequences from thedisclosed chimpanzee nucleic acid sequences and insertion of sequencesderived other DNA sequences that are useful for transgene insertion,expression or other genetic manipulations. Accordingly, the recombinantchimpanzee adenoviruses described herein may contain adenoviralsequences derived from one or more chimpanzee adenoviruses, or sequencesfrom a chimpanzee adenovirus and from a human adenovirus. Suitablepolynucleotide sequences can be produced recombinantly, synthetically orisolated from natural sources. Adenoviral sequences suitable for use inparticular aspects of the invention include sequences which lackneutralizing B-cell epitopes that are cross-reactive with common humanserotypes.

At a minimum, the recombinant chimpanzee adenovirus (e.g., vector) ofthe invention contain the chimpanzee adenovirus cis-acting elementsnecessary for replication and virion encapsidation, in combination withat least one immunogen expression cassette. Typically, the cis-actingelements flank the expression cassette which comprises a transgene thatencodes at least one antigen. More specifically, the vectors of theinvention contain the requisite cis-acting 5′ inverted terminal repeat(ITR) sequences of the adenoviruses (which function as origins ofreplication), 3′ ITR sequences, packaging/enhancer domains, and anucleotide sequence encoding a heterologous molecule. Regardless ofwhether the recombinant vector comprises only the minimal adenoviralsequences or an entire adenoviral genome with only functional deletionsin particular genes (e.g., the E1 and/or E3 or E4 regions), the vectorsof the invention comprise a chimpanzee adenovirus capsid.

Generally, speaking the adenoviral vectors disclosed herein comprise areplication-defective adenoviral genome, wherein the adenoviral genomedoes not have a functional E1 gene, and an immunogen expression cassettewhich comprises: a) a nucleic acid encoding at least one immunogenagainst which an immune response is desired; and b) a heterologous(i.e., with respect to the adenoviral sequence) promoter operativelylinked to the nucleic acid sequence encoding the immunogen(s); and atranscription terminator.

More specifically, the invention provides replication-defective vectorsthat consist of a recombinant adenoviral genome that is devoid of atleast one early gene selected from the group consisting of E1, E2, E3,and E4. In one embodiment, a replication-defective vector is prepared byreplacing, or disrupting, the E1 gene of one of the adenoviral isolatesdisclosed herein (e.g., ChAd3, ChAd6, ChAd4, ChAd5, ChAd7, ChAd9,ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd8, ChAd22, ChAd24,ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 or ChAd82) withan immunogen expression cassette. For example, a vector can be preparedby deleting/disrupting the E1 gene of ChAd 3 (SEQ ID NO:1) or ChA6 (SEQID NOS: 2). Alternatively, a replication-defective vector can beprepared from any one of the other adenovirus isolates disclosed herein,including ChAd3, ChAd6, ChAd4, ChAd5, ChAd7, ChAd9, ChAd10, ChAd11,Chad16, Chad17, ChAd19, ChAd8, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31,ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 or ChAd20. In otherembodiments, replication-defective vectors of the invention comprises anadenoviral genome derived from one of the ChAds disclosed herein thathas been optionally engineered to lack a functional E3 gene. It is to beunderstood that the chimpanzee adenoviral sequences disclosed herein canbe rendered replication-defective by either completely removing an earlygene or by rendering the gene inoperative or nonfunctional.

It is to be understood that the invention encompasses vectors that arecharacterized as having modifications, such as a “functional deletion”which destroys the ability of the adenovirus to express one or moreselected gene products. The phrase “functional deletion” as used hereinbroadly encompasses modifications that have the effect of rendering aparticular gene product nonfunctional. Generally speaking, functionaldeletions take the form of a partial or total deletion of an adenoviralgene. However, one of skill in the art will readily acknowledge thatother manipulations, including but not limited to making a modificationwhich introduces a frame shift mutation, will also achieve a functionaldeletion. For example, the recombinant chimpanzee adenoviral vectors ofthe invention can be rendered replication-defective by introducing amodification that is designed to interfere with, or to functionallydelete, the ability of the virus to express adenoviral E1A and/or E1B.

It is well-known that replication-defective adenoviral vectors can beobtained by introducing a modification that is designed to interferewith, or to functionally delete the expression of one or more genes fromthe group of E2 genes. More in detail, a replication-defective vectorcan be constructed by inactivating the polymerase gene, or thepre-terminal protein gene or the DNA binding protein gene. Moreoverdeletion or inactivation of genes expressed by E4 region is analternative strategy to construct replication-defective chimp Advectors. Early gene deletion or inactivation can be combined in order toproduce more attenuated vectors. Alternatively, replication-defectiveChAd vectors can also comprise additional modifications in other viralgenes, such as the late genes L1 through L5. In addition, noveladenoviral vaccine carriers can be generated by combining hexon andfiber genes obtained from different serotypes. The utilization of ahexon and fiber gene shuffling strategy will also allow an investigatorto change the biological properties of a ChAd and facilitate theproduction of vectors with a different tropism or with new serologicalcharacteristics.

It is to be understood that the present invention encompassesrecombinant adenoviral vectors comprising deletions of entire genes orportions thereof which effectively destroy the biological activity ofthe modified gene either alone or in any combination. For example,recombinant simian adenoviruses can be constructed which have afunctional deletion of the genes expressed by E4 region, although asshown herein it may be desirable to introduce the heterologous Ad5 E4sequence into the vector in combination with the functional deletion ofan E1 gene. Alternatively, the function of the adenoviral delayed earlyE3 gene may be eliminated; however because the function of E3 is notnecessary for the production of a recombinant adenoviral particle it isnot necessary to replace this gene product in order to produce arecombinant that is capable of packaging a virus useful in theinvention.

In one embodiment of this invention, the replication-defectiveadenoviral vector used is a chimpanzee subgroup C adenovirus containingdeletions in E1 and optionally in E3. For example, for ChAd3, a suitableE1 deletion/disruption can be introduced in the region from by 460 to by3542 (with reference to SEQ ID NO: 1). For ChAd6, a suitable E1deletion/disruption can be introduced in the region from by 457 to by3425 (with reference to SEQ ID NO: 2). For CV32, the E1 deletion ispreferably from by 456 to by 3416 (with reference to SEQ ID NO: 3); forCV33, the E1 deletion is preferably from by 456 to by 3425 (withreference to SEQ ID NO: 4) and for CV23, the E1 deletion is preferablyfrom by 456 to by 3415 (with reference to SEQ ID NO: 5). E3 deletionsfor CV32 and CV33 are preferably from by 27446 to by 31911 (withreference to SEQ ID NO: 3); from by 27146 to by 31609 (with reference toSEQ ID NO: 4) respectively. Those of skill in the art can easilydetermine the equivalent sequences for other chimpanzee isolates basedon sequence homologies and multiple sequence alignments.

One of skill in the art will readily acknowledge that in order toconstruct an E1-deleted adenoviral vector a number of decisions must bemade regarding the structure of the vector backbone and the compositionof the nucleic acid sequence comprising the transgene. For example, aninvestigator must determine if the size of the E1 deletion willaccommodate the size of the transgene. If not, then additional deletionswill have to be introduced into the backbone of the vector.

The nucleic acid sequence embodying the transgene can be a gene, or afunctional part of a gene and will typically exist in the form of anexpression cassette. Typically a gene expression cassette includes: (a)nucleic acid encoding a protein or antigen of interest; (b) aheterologous promoter operatively linked to the nucleic acid encodingthe protein; and (c) a transcription termination signal. The nucleicacid can be DNA and/or RNA, can be double or single stranded. Thenucleic acid can be codon-optimized for expression in the desired host(e.g., a mammalian host).

Decisions must also be made regarding the site within the backbone wherethe transgene will be introduced and the orientation of the transgene.More specifically, the transgene can be inserted in an E1 parallel(transcribed 5′ to 3′) or anti-parallel (transcribed in a 3′ to 5′direction relative to the vector backbone) orientation. In addition,appropriate transcriptional regulatory elements that are capable ofdirecting expression of the transgene in the mammalian host cells thatthe vector is being prepared for use as a vaccine carrier in need to beidentified and operatively linked to the transgene. “Operatively linked”sequences include both expression control sequences that are contiguouswith the nucleic acid sequences that they regulate and regulatorysequences that act in trans, or at a distance to control the regulatednucleic acid sequence.

Regulatory sequences include: appropriate expression control sequences,such as transcription initiation, termination, enhancer and promotersequences; efficient RNA processing signals, such as splicing andpolyadenylation signals; sequences that enhance translation efficiency(e.g., Kozak consensus sequences); sequences that enhance proteinstability, and optionally sequences that promote protein secretion.Selection of these and other common vector elements are conventional andmany suitable sequences are well known to those of skill in the art(see, e.g., Sambrook et al, and references cited therein at, forexample, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, 1989).

In specific embodiments, the promoter is a heterologous promoter (i.e.,with respect to the adenovirus sequences) which is recognized by aneukaryotic RNA polymerase. In a preferred embodiment, the promoter is a“strong” or “efficient” promoter. An example of a strong promoter is theimmediate early human cytomegalovirus promoter (Chapman et al, 1991Nucl. Acids Res 19:3979-3986, which is incorporated by reference). Thehuman CMV promoter can be used without (CMV) or with the intron Asequence (CMV-intA), although those skilled in the art will recognizethat any of a number of other known promoters, such as the strongimmunoglobulin, or other eukaryotic gene promoters may be used,including the EF1 alpha promoter, the murine CMV promoter, Rous sarcomavirus (RSV) promoter, SV40 early/late promoters and the beta-actinpromoter.

Further examples of promoters that can be used in the present inventionare the strong immunoglobulin promoter, the EF1 alpha promoter, themurine CMV promoter, the Rous Sarcoma Virus promoter, the SV40early/late promoters and the beta actin promoter, albeit those of skillin the art can appreciate that any promoter capable of effectingexpression in the intended host can be used in accordance with themethods of the present invention. The promoter may comprise aregulatable sequence such as the Tet operator sequence. Sequences suchas these that offer the potential for regulation of transcription andexpression are useful in instances where repression of genetranscription is sought.

Suitable gene expression cassettes will also comprise a transcriptiontermination sequence. A preferred transcriptional terminator is thebovine growth hormone terminator. The promoter/transcription terminationcombination of CMVintA-BGH terminator is particularly preferred althoughother promoter/terminator combinations may also be used. As shownherein, the bovine growth hormone termination/polyadenylation signal(bGHpA) or short synthetic polyA signal (SPA) of 50 nucleotides inlength defined as follows:

(SEQ ID NO: 26) AATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTG.Generally speaking, exemplify suitable termination sequences. The polyAsignal is inserted following the nucleic acid sequence which comprisesthe transgene and before the 3′ adenovirus ITR sequence.

The recombinant adenoviral vectors described herein may containadenoviral sequences derived from one or more strain of adenovirus.Suitable sequences may be obtained from natural sources, producedrecombinantly, synthetically, or by other genetic engineering orchemical methods. In a particular embodiment, the recombinant chimpanzeeadenovirus is a chimeric recombinant comprising non-chimpanzeeadenoviral polynucleotide sequences. Suitable non-chimpanzee adenoviralsequences can be obtained from human adenoviral strains. For example,the native E4 region can be replaced by hAd5 E4 (Ad5 nt 32816 to nt35619) or by Ad5E4orf6 (Ad5 nt 33193 to nt 34077) (Ad5 GenBank AccessionNo: M73260).

Generally speaking, the immunogen (antigenic molecule) delivered by therecombinant adenoviral vector of the invention comprises a polypeptide,protein, or enzyme product which is encoded by a transgene incombination with a nucleotide sequence which provides the necessaryregulatory sequences to direct transcription and/or translation of theencoded product in a host cell. The composition of the transgene dependsupon the intended use of the vector. For example, if the immunogeniccomposition is being designed to elicit an antibody response or acell-mediated immune response in a mammalian host which is specific foran infectious agent, then it is appropriate to utilize a nucleic acidsequence encoding at least one immunogenic product that is predicted toconfer pathogen-specific immunity to the recipient. Alternatively, ifthe composition is being prepared for use as a cancer vaccine, asuitable transgene may comprise an immunogenic portion of aself-antigen, such as a TAA, which has been selected with the goal ofeliciting a protective immune response of sufficient potency to bothbreak host tolerance to a particular TAA and to elicit a long-lived(e.g., memory) response that will be sufficient to prevent theinitiation of cancer or to prevent tumor progression. Accordingly,suitable immunogenic gene products may be obtained from a wide varietyof pathogenic agents (such as, but not limited to viruses, parasites,bacteria and fungi) that infect mammalian hosts, or from a cancer ortumor cell. Although, the invention is illustrated herein with aparticular set of test immunogens it is to be understood that theinvention is not limited to the use of the antigens exemplified herein.More specifically, the invention contemplates the use of bothheterologous and self-antigens as immunogens, including but not limitedto TAAs.

In one embodiment, the invention provides an immunogenic composition(e.g., a vaccine) for inducing an immune response against antigens(i.e., immunogens) expressed by an infectious agent. For example, it isdesirable to elicit an immune response against a virus infecting humansand/or non-human animal species. Examples of virus families againstwhich a prophylactic and/or therapeutic immune response would bedesirable include the Picornaviridae family which includes six differentgenera such as Aphtovirus, Cardiovirus, Enterovirus, Hepatovirus,Parechovirus, Rhinovirus. Examples of Picornavirus against which animmune response would be desirable are: Foot-and-mouth disease viruses,Encephalomyocarditis viruses, Polioviruses, Coxackieviruses, Humanhepatitis A virus, Human parechoviruses, Rhinoviruses. Caliciviridaefamily includes different genera associated with epidemicgastroenteritis in humans caused by the Norwalk group of viruses andother syndromes in animals like the hemorrhagic disease in rabbitsassociated with rabbit hemorrhagic disease virus or respiratory diseasein cats caused by feline calicivirus.

Another family of viruses, against which it may be desirable to elicitan immune response is the Astroviridae which comprises viruses isolatedfrom humans as well as many different animal species. Human astrovirusesare associated with gastroenteritis and young children diarrhea.Alternatively, it may be desirable to confer mammalian hosts withimmunity to members of the Togaviridae family of viruses which comprisestwo genera: alphavirus and rubivirus. Alphaviruses are associated withhuman and veterinary diseases such as arthritis (i.e. Chikungunya virus,Sindbis virus) or encephalitis (i.e. Eastern Equine Encephalitis Virus,Western Equine Encephalitis Virus).

Rubella virus provides an alternative viral target against which is theonly member of the Rubivirus genus is responsible for outbreaks of amild exanthematic disease associated with fever and lymphoadenopathy.Rubella virus infection is also associated with fetus abnormalities whenacquired by mother during in early pregnancy. Flaviviridae is anothervirus family consisting of three genera: the flaviviruses, thepestiviruses and the hepaciviruses that includes important human as wellas animal pathogens. Many of the flavivirus genus members arearthropod-borne human pathogens causing a variety of diseases includingfever, encephalitis and hemorrhagic fevers. Dengue Fever Viruses, YellowFever Virus, Japanese Encephalitis Virus, West Nile Fever Virus,Tick-borne Encephalitis Virus are pathogens of major global concern orof regional (endemic) concern. Pestivirus genus includes animalpathogens of major economic importance such as Bovine Viral DiarrheaVirus, Classical Swine Fever Virus, Border Disease Virus. Hepatitis CVirus is the only member of the Hepacivirus genus responsible for acuteand chronic hepatitis. HCV proteins expressed by a recombinantadenovirus can elicit a protective as well as therapeutic immuneresponse limiting the consequences of a viral infection affecting 170million people worldwide.

Alternatively, antigens derived from members of the Coronaviridae familycan be expressed by recombinant adenovirus vectors in order to obtainprotection against infection. Protection against the severe acuterespiratory syndrome coronavirus (SARS-Co Virus) can be obtained byimmunizing with one or more chimpanzee adenovirus chosen from the groupincluding ChAd3, 4, 5, 6, 7, 9, 10, 11, 16, 17, 19, 20, ChAd8, ChAd22,ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 andChAd82 expressing one or more SARS-CoV protein including withoutlimitations nucleocapsid (N) protein, polymerase (P) protein, membrane(M) glycoprotein, spike (S) glycoprotein, small envelope (E) protein orany other polypeptide expressed by the virus. Rhabdoviridae familymembers including rabies virus can be target of recombinant vaccineexpressing viral proteins.

Other possible targets include the Filoviridae family comprisingEbola-like viruses and Marburg-like viruses genera, that is responsibleof outbreaks of severe hemorrhagic fever; the Paramyxoviridae familycomprising some of the most prevalent virus known in humans likemeasles, respiratory syncytial, parainfluenza viruses and viruses ofveterinary interest like Newcastle disease and rinderpest viruses; theOrthomyxoviridae family including Influenza A, B, C viruses;Bunyaviridae family mainly transmitted by arthropod to vertebrate hostscomprising important human pathogens like Rift valley fever, Sin Nombre,Hantaan, Puumala viruses; Arenaviridae family comprising Lymphocyticchoriomeningitis, Lassa fever, Argentine Hemorragic fever, BolivianHemorragic fever viruses; Bornaviridae family comprising viruses causingcentral nervous system diseases mainly in horses and sheep; Reoviridaefamily including rotaviruses, the most important cause of severediarrheal illness in infants and young children worldwide, orbivirusesthat can affect both humans and other mammals (bluetongue, epizootichemorrhagic disease viruses); Retroviridae family, a large group ofviruses comprising important human pathogens like human immunodeficiencyvirus 1 and 2 (HIV-1 and HIV-2) and human t-cell leukemia virus type 1and 2 (HTLV 1 and 2) as well as non-human lentivirus such as Maedi/Visnaviruses affecting sheep and goats, Equine infectious anemia virusaffecting horses, bovine immunodeficiency virus affecting cattle, felineimmunodeficiency virus affecting cats; Polyomaviridae family groupssmall DNA oncogenic viruses, prototype viruses are polyoma and SV40infecting mouse and rhesus monkey respectively, (BK and JC virusesclosely related to SV40 were isolated from human patients);Papillomaviridae family consists of a group of DNA viruses infectinghigher vertebrates including humans generating warts and condylomas.Papilloma viral infection is associated with the development of cancerin both humans and animals. Human papilloma viruses are associated withcervical cancer, vaginal cancer and skin cancer. The herpesviridaefamilies includes subfamilies in which are classified a number ofimportant pathogens for humans and other mammals. Suitable sources ofantigens can be but are not limited to herpes simplex viruses 1 and 2,varicella-zoster virus, Epstein-Barr virus, Cytomegalovirus, humanherpesviruses 6A, 6B and 7, Kaposi's sarcoma-associated herpesvirus.Further suitable source of antigens are members of the Poxyiridae familylike Monkeypox virus, Molluscum contagiusum virus, smallpox virus;Hepatitis B virus, the prototype member of the hepadnaviridae family aswell as other virus causing acute and/or chronic hepatitis likehepatitis delta virus, hepatitis E virus.

The adenoviral vectors of the present invention are also suitable forthe preparation of immunogenic compositions designed to stimulate animmune response in humans or animals against protein expressed bynon-viral pathogens including bacteria, fungi, parasites pathogens. Forexample, the vectors disclosed herein can be used to prepare vaccinesagainst, but not limited to: Staphylococcus aureus, Streptococcuspyogenes, Streptococcus pneumoniae, Vibrio cholerae, Clostridium tetani,Neisseria meningitis, Corynebacterium diphteriae, Mycobacteriatuberculosis and leprae, Listeria monocytogenes, and Legionellapneumofila. Examples of fungi and mammalian parasites for which it maybe desirable to prepare prophylactic or therapeutic vaccines include:Candida albicans, Aspergillus fumigatus, Histoplasma capsulatum,Plasmodium malariae, Leishmania major, Trypanosome cruzi and brucei,Schistosoma haematobium, mansoni and japonicum; Entamoeba histolytica,and numerous species of Filaria known to be responsible for humanfilariasis.

Cancer typically involves the deregulation of genes that encodepolypeptides which contribute to maintaining cell cycle or controllingcell proliferation (e.g., growth factors, oncogenes, receptors and tumorsuppressors). The products of many of the genes implicated in cancer areexpressed on the surface of a wide variety of tumor cells. A variety oftumor antigens that may be recognized by T and B lymphocytes have beenidentified in human and animal cancer. The vast majority of humantumor-associated antigens (TAAs) that are suitable for use in ananticancer vaccine trial are described as “self-antigens” due to thefact that in addition to being expressed on tumor cells they also areexpressed on normal tissue and/or during fetal developmentImmunotolerance of the target population to TAAs may explain why manycancer vaccines have proven to be ineffective.

Tumor antigens can be produced by oncogenic mutants of normal cellulargenes altered proto-oncogenes or tumor suppressor genes such as Ras, p53or Bcr-Abl protein are examples of altered cellular proteins that canstimulate T/B cell response. Tumor antigens can be normal cellularproteins that are overexpressed in tumor cells (tyrosinase, GP100, MARTare normally expressed at low levels in melanocytes and overexpressed inmelanoma) or aberrantly expressed in tumor cells (MAGE, BAGE, GAGEexpressed in melanomas and many carcinomas but normally expressed in thetestis and placenta). Tumor antigens can be products of oncogenicviruses: papillomavirus E6 and E7 proteins expressed by cervicalcarcinomas; EBV EBNA-1 protein produced by EBV+lymphomas andnasopharyngeal carcinomas; SV40 T antigen in SV40 induced experimentaltumors. Oncofetal antigens are expressed to high levels on cancer cellsand in normal developing (fetal) tissues but not in adult tissues.Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) are examplesof well characterized oncofetal antigens.

Recent evidence supports the existence of TAAs that are capable ofeliciting an immune response, thus making this class of antigenssuitable immunogens for vaccine therapy. However, as a class of antigensTAAs are notoriously poor immunogens and T cells that are highlyspecific for TAAs are either deleted or anergized during T-celldevelopment. Accordingly, there is an expectation that the immuneresponse of a tumor-bearing host to a particular TAA will be extremelyweak. Because of the inherent need to break host tolerance to a targetTAA experimental clinical vaccine studies are particularly focused ondeveloping immunization strategies that will enhance TAA-specific T-cellresponses. Generally, speaking an effective cancer vaccine must bothovercome immunotolerance and enhance host's immune response to a levelthat is preventative and/or protective. Anti-tumor effects in manyexperimental vaccine studies have been correlated with T-cell responsesto TAAs.

In an alternative embodiment, the invention contemplates an immunogeniccomposition (e.g., a cancer vaccine) which can be used to induce animmune response against tumor antigens. A suitable composition wouldcontain a recombinant chimpanzee adenovirus comprising nucleic acidsequence encoding a tumor antigen and a physiologically acceptablecarrier. In a particular embodiment, the coding sequence element of thecassette may encode a single immunogen, such as an immunogenic peptidesequence derived from a self-antigen, such as a tumor-associatedantigen. In some embodiments, the nucleic acid sequence encoding theimmunogen (i.e., the transgene) may be codon optimized for expression ina particular mammalian species. In other embodiments, the codingsequence may encode more than one immunogen, such as one or more codonoptimized tumor antigens. For example, a cancer vaccine utilizing thedisclosed adenoviral vectors may encode a combination of self-antigenssuch as: HER2/neu, CEA, Hepcam, PSA, PSMA, Telomerase, gp100,Melan-A/MART-1, Muc-1, NY-ESO-1, Survivin, Stromelysin 3, Tyrosinase,MAGE3, CML68, CML66, OY-TES-1, SSX-2, SART-1, SART-2, SART-3, NY-CO-58,NY-BR-62, hKLP2, VEGF.

Development of an effective cancer vaccine requires the identificationof a strategy that will elicit antigen-specific immunity in vaccinatedpatients and the generation of an immune response that will persistafter active immunization has ended. The success of the strategy willdepend on whether a measurable immune response directed against a targetantigen will correlate with protection against cancer occurrence orrelapse. The effector mechanisms of both cell-mediated immunity andhumoral immunity have been show to kill tumor cells. However, data fromexperimental systems suggest that antigen-specific T cells represent themost powerful immunologic mechanism for the elimination of tumor cells.Recognition of tumor-specific antigens (e.g., TAAs) by effector T-cellsis predicted to allow the TAA to function as a tumor-rejection antigen.Published studies suggest that stimulation of CD8′ and CD4 helper T-cellresponses are important for achieving optimal tumor clearance((Greenberg, P. D. (1991) Adv. Immunol. 49: 281-355; Pardoll, D. M. etal. (1998) Curr. Opin. Immunol. 10: 588-94). Clinical response (i.e.,efficacy) has been associated with increases in interferon γ-secretingcytotoxic T cells. The advent of assays, such as the ELISPOT assay usedherein, to demonstrate the efficacy of the instant vaccine carriers,allows investigators to measure T-cell responses to vaccination regimensand thereby facilitates the development of cancer vaccines.

Cancer vaccines can be either prophylactic or therapeutic. The generalassumption underlying the prophylactic use of cancer vaccines is thatTAAs are extremely weak immunogens or functionally nonimmunogenic intumor-bearing subjects. More specifically, in the field of cancerimmunology, vaccines can be used as immunotherapy in patients afflictedwith cancer. Accordingly, cancer vaccines can be designed to elicit animmune response that is that is directed against a TAA that is expressedby a pre-existing tumor or malignancy. Thus, in particular embodiments,therapeutic cancer vaccines are intended for use in tumor-bearingpatients who have developed resistance to conventional regimens oftreatment or who have a high probability of developing a recurrencefollowing conventional treatment.

The high immunogenicity of adenoviruses, make adenoviral vectorsparticularly good candidates for use in the context of a vaccine carrierdesigned to break host tolerance to a self-antigen. The phenomenon ofepitope or determinant spreading, which was first described inautoimmune diseases, has been associated with both MHC class I- and MHCclass II-restricted responses and correlated to the development ofHER-2/neu protein-specific T-cell immunity. Epitope spreading representsthe generation of an immune response to a particular portion of animmunogenic protein followed by the natural spread of immunity to otherantigenic determinants present on the same protein. For example, Disiset al. observed epitope spreading in 84% of patients afflicted withHER-2/neu overexpressing malignancies who were administered vaccinescomprising peptides derived from potential T-helper epitopes of theHER-2 protein mixed with granulocyte-macrophage colony stimulatingfactor (J. Clin. Oncol. (2002) 20(11): 2624-2632). Importantly, epitopespreading was correlated with the generation of a HER-2/neu proteindomain response and suggests that immunization effectively circumventedimmunologic tolerance.

TAAs that are suitable for use in the disclosed adenoviral vectors andmethods as a target for a cancer vaccine should possess a number ofcharacteristics. For example, a target TAA must have a favorableexpression profile, meaning that it should be preferentially expressedor overexpressed in the tumor or malignant tissue as compared withnormal tissue. In addition, because TAAs that play a role intumorigenesis are more likely to be retained during the different stagesof cancer progression, a suitable target TAA should also preservedthroughout tumor progression and metastases. Suitable target TAAs shouldalso be expressed homogenously within the tumor. Third, suitable targetTAAs must not be subject to absolute immunologic tolerance. Morespecifically, there should be some evidence that T cells which can bothrecognize and respond to the TAA of interest have not been entirelydeleted from the host's T-cell repertoire (Berinstein, N. L., J. Clin.Oncol. 29(8): 2197 (2002).

Carcinoembryonic antigen (CEA) has many characteristics which make it anattractive TAA for use as a target antigen for an anticancer vaccine. Itis a member of the Ig superfamily which is characterized by a favorableexpression pattern. It is expressed in more than 50% of all humancancers and has been implicated in the tumorigenesis process, whichsuggests that its expression may be selected and conserved throughoutcancer progression. In addition, it has been established thatimmunologic tolerance to CEA is not absolute. Published studiesestablish that human T cells can recognize, become activated to, andlyse cancer cells that express CEA (Berinstein, N. L., J. Clin. Oncol.29(8): 2197 (2002). For example, the immunization of patients withrecombinant vaccinia virus expressing CEA, combined with subsequentpeptide-based in vitro stimulations, generated CD8+ MHC-restricted CTLscapable of lysing autologous tumors (Tsang, K. Y. et al. J. Natl. CancerInst., (1995) 87:982-990). Alternatively, immunization of colorectalcarcinoma patients after surgery with recombinant CEA was reported toinduce weak antibody and cellular responses to recombinant CEA (Samanci,A., et al. (1998) Cancer Immunol. Immunother. 47: 131-142.) Further, theadministration of anti-CEA anti-idiotypic antibody to patients diagnosedwith colorectal cancer generated anti-CEA antibodies andidiotype-specific T-cell proliferation (Foon, L, A. et al. (1995) J.Clin. Invest., 96: 334-342). The literature also indicates thattolerance to CEA in cancer patients can be overcome with severaldifferent vaccination approaches (i.e., vaccination with recombinant CEAor recombinant orthopox or avipox-CEA viruses, administration ofanti-idiotype antibodies, pulsing dendritic cells with CEA agonistepitopes).

CEA is an oncofetal glycoprotein that is expressed in normal fetal colonand to a much lesser extent in normal colonic mucosa. It is alsooverexpressed in the vast majority of adenocarcinomas, particularlythose of the colon, pancreas, breast, lung, rectum and stomach. Manycolorectal cancers and some carcinomas produce high levels of CEA thatare measurable in sera, which makes it one of the most widely usedserological markers of malignancy, especially in patients withcolorectal cancer.

A second TAA which provides a suitable immunogen for use in thecompositions and methods of the invention is product of the HER2/erb-2(also called neu) proto-oncogene. Like, CEA, HER2/neu has a favorableexpression pattern and is not subject to absolute tolerance. Morespecifically, low levels of expression of the HER2/neu transcript, andthe 185 kD polypeptide product, are detected in normal adult epithelialcells of various tissues, including the skin and breast, and tissues ofthe gastrointestinal, reproductive, and urinary tracts; higher levels ofexpression are detected in the corresponding fetal tissues duringembryonic development (Press et al., Oncogene 5: 953-962 (1990). Severallines of evidence suggest a link between the amplification of HER-2 andneoplastic transformation in human breast, lung, prostate, ovarian,endometrial and colorectal tumors (Disis and Cheever, Adv. CancerResearch 71: 343-371 (1997). Generally speaking, overexpression ofHER2/neu correlates with a poor prognosis and a higher relapse rate forcancer patients (Slamon et al., Science 244: 707-712 (1989). Thus, avaccine specific for the HER-2/neu protein could have wide applicationand utility in the prevention of disease recurrence in many differenthuman malignancies.

HER2/neu encodes a transmembrane glycoprotein possessing intrinsictyrosine kinase activity and displaying extensive homology to theepidermal growth factor (EGF) receptor (Akiyama, T et al., (1986)Science 232: 1644-1646). One of the first clinical studies whichutilized HER2 as target for cancer immunotherapy employed theHER-2-specific monoclonal antibody Herceptin for the treatment of breastcancer (Goldenberg M M (1999) Clin. Ther. 21: 309-318). This led tosubsequent efforts which focused on the use of HER-2 as a target for theT-cell arm of the immune system to elicit effective antitumor responses,including the use of recombinant fusion proteins comprising HER-2domains to activate autologous antigen presenting cells. Publishedreports establish that numerous cancer patients afflicted withneu-expressing mammary and ovarian cancers mount immune responses (e.g.,produce antigen-specific antibody and T-cells) against the proteinproduct of the HER2/neu oncogene.

Assembly of the recombinant adenoviral sequences, transgene and othervector elements into various intermediate plasmids and shuttle vectors,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques as described instandard textbooks that are well known to those of skill in the art(Sambrook et al, Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Suchtechniques include, but are not limited to conventional cDNA cloningtechniques, use of overlapping oligonucleotide sequences derived fromthe adenoviral genome, homologous recombination, polymerase chainreaction, standard transfection techniques, plaquing of viruses in agaroverlay and other related methodologies.

To assist in preparation of polynucleotides in prokaryotic cells, aplasmid version of the adenovirus vector is often prepared (adenoviruspre-plasmid). The adenovirus pre-plasmid contains an adenoviral portionand a plasmid portion. The adenoviral portion is essentially the same asthe adenoviral portion contained in the adenoviral vectors of theinvention (containing adenoviral sequences with non-functional ordeleted E1 and optionally E3 regions) and an immunogen expressioncassette, flanked by convenient restriction sites.

The plasmid portion of the adenovirus pre-plasmid often contains anantibiotic resistance marker under transcriptional control of aprokaryotic promoter so that expression of the antibiotic does not occurin eukaryotic cells. Ampicillin resistance genes, neomycin resistancegenes and other pharmaceutically acceptable antibiotic resistancemarkers may be used. To aid in the high level production of thepolynucleotide by fermentation in prokaryotic organisms, it isadvantageous for the adenovirus pre-plasmid to contain a prokaryoticorigin of replication and be of high copy number. A number ofcommercially available prokaryotic cloning vectors provide thesebenefits. It is desirable to remove non-essential DNA sequences. It isalso desirable that the vectors not be able to replicate in eukaryoticcells. This minimizes the risk of integration of polynucleotide vaccinesequences into the recipients' genome. Tissue-specific promoters orenhancers may be used whenever it is desirable to limit expression ofthe polynucleotide to a particular tissue type.

Adenovirus pre-plasmids (plasmids comprising the genome of thereplication-defective adenovirus with desired deletions and insertions)can be generated by homologous recombination using adenovirus backbonesDNA and an appropriate shuttle vector (designed to target-in specificdeletions and incorporate desired restriction sites into the resultantplasmid). Shuttle vectors of use in this process can be generated usinggeneral methods widely understood and appreciated in the art, e.g., PCRof the adenoviral terminal ends taking into account the desireddeletions, and the sequential cloning of the respective segments into anappropriate cloning plasmid. The adenoviral pre-plasmid can then bedigested and transfected into the complementing cell line via calciumphosphate co-precipitation or other suitable means. Virus replicationand amplification then occurs, a phenomenon made evident by notablecytopathic effect. Infected cells and media are then harvested afterviral replication is complete (generally, 7-10 days post-transfection).

Generally speaking, following the construction and assembly of thedesired adenovirus pre-plasmids, adenovirus pre-plasmids are rescuedinto virus by transfecting an adenoviral E1-expressing human cell line.Complementation between the packaging cell line and the viral genes ofthe vector permits the adenovirus-transgene sequences in the vector tobe replicated and packaged into virion capsids, resulting in theproduction of recombinant adenoviruses. The resulting viruses may beisolated and purified by any of a variety of methods known to those ofskill in the art for use in the methods of the invention.

It will be readily apparent to those of skill in the art that when oneor more selected deletions of chimpanzee adenoviral genes are introducedinto a viral vector, the function of the deleted gene product can besupplied during the production process by sequences present in theproduction cell line. Thus, the function of the manipulated genes can beprovided by a permanently transformed cell line that is characterized bysome or all of the adenoviral functions which are required for packagingbut which are not functional in the vector (e.g., any of E1A, E1B, E2A,E2B E4). Alternatively, the requisite adenoviral functions can beprovided to a suitable packaging cell line by infecting or transientlytransfecting a suitable cell with a construct comprising the requisitegene to provide the function.

Accordingly, the present invention also provides a method of producingchimpanzee adenoviral vectors in E1-expressing human cell lines. Morespecifically, the disclosed vectors can be propagated in an E1complementing cell lines, including the known cell lines 293 andPER.C6™. Both these cell lines express the adenoviral E1 gene product.PER.C6™ is described in WO 97/00326, published Jan. 3, 1997, which ishereby incorporated by reference. It is a primary human retinoblast cellline transduced with an E1 gene segment that complements the productionof replication deficient first generation adenoviruses, but is designedto prevent generation of replication competent adenovirus by homologousrecombination. 293 cells are described in Graham et al (1977) J. Gen.Virol 36:59-72, which is also hereby incorporated by reference. One ofskill in the art will recognize the term “first generation adenovirus”refers to a replication deficient adenovirus which has either anon-functional or deleted E1 region, and optionally a non-functional ordeleted E3 region.

Batches of replication-defective adenoviral vectors that are intendedfor use as a vaccine composition in a clinical trial should be proven tobe free of RCA (Fallaux, F. J. et al (1998) Human Gene Therapy,9:1909-1917). In practice, this is a labor intensive process whichrequires establishing and utilizing an expensive screening program. Oneof skill in the art will acknowledge that a high frequency of RCAgeneration not only results in a high failure rate for the batchesproduced, but also severely limits scale-up efforts. Elimination ofsequence homology between the nucleotide sequence of the vector and theadenoviral sequences present in the genome of the helperproduction/packaging cell line should eliminate the possibility ofproducing batches of vector that are contaminated with RCAs produced byhomologous recombination.

Typically, recombinant replication-defective adenoviral vectors arepropagated in cell lines that provide E1 gene products in trans.Supplementation of the essential E1 gene products in trans is veryeffective when the vectors are from the same or a very similar serotype.For example, it is well-known that E1-deleted (i.e. ΔE1) group Cserotype (Ad2 and Ad5) vectors, can be propagated in 293 or PER.C6 cellswhich contain and express the Ad5 E1 region. However, it has beenobserved that Ad5 E1 sequences present in the 293 and PER.C6 productioncells may not always fully complement the replication of non-group Cserotypes. Accordingly, E1-deleted serotypes outside of subgroup C, forexample those from subgroups A, B, D, E, and F may replicate with alower efficiency respect to the corresponding wt virus or may notreplicate at all in 293 or PER.C6 cells. This may be due to theinability of the Ad5 (group C) E1B 55K gene product to establish afunctional interaction with the E4 orf6 gene product of the non-group Cserotypes.

The decrease in replication efficiency in cells expressing Ad5 E1 isvariable considering vectors of different subgroups. While ΔE1 vectorsderiving from subgroup D and E adenovirus can be rescued and propagatedin 293 and Per.C6™ cells with variable efficiency, the propagation ΔE1vectors of subgroup B is completely impaired (Vogels R, et. al. (2003)August Replication-deficient human adenovirus type 35 vectors for genetransfer and vaccination: efficient human cell infection and bypass ofpreexisting adenovirus immunity. J Virol. 77 (15):8263-71).

Although the interaction between Ad5 E1b 55k and vector-expressing E4orf6 protein is conserved within members of the same subgroup, it may benot sufficiently stable when E4 orf6 protein of a non-C serotype isexpressed. This inefficient or unstable formation of E1B-55K/E4-orf6complex lead to an absent of reduced propagation of the ΔE1 vector.Accordingly, it has been empirically determined that in order tosuccessfully and efficiently rescue recombinant adenovirus of groupBserotypes, a cell line expressing the E1 region of the serotype ofinterest may need to be generated. In cells expressing Ad5E1 like 293 orPer.C6™, the expression can be limited to E1b 55K protein.Alternatively, a suitable Ad5E1-expressing cell lines could be modifiedto express the entire Ad5 E4 region (or E4 orf6 only) in addition toAd5E1. The generation of cell lines expressing both Ad5 E1 and orf6 areuseful in complementing alternative adenovirus serotypes; see, e.g.,Abrahamsen et al., 1997 J. Virol. 8946-8951. The incorporation of E4(orf6) into Ad5 complementing cell lines, is known, as is the generationof serotype-specific cell lines providing a serotype-specific E1 geneproduct(s) in trans. Alternatively, the efficiency of non-group C vectorpropagation may be improved by modification of the viral backbone bysubstituting the native E4 region with Ad5 orf6. Similar results can beachieved by substituting the only the native orf6 with orf6 derivingfrom Ad5 or other subgroup C viruses (Ad1, Ad2, Ad6). U.S. Pat. No.5,849,561 discloses complementation of an E1-deleted non-group Cadenovirus vector in an Ads-E1 complementing cell line which alsoexpresses portions of the Ad5-E4 gene.

U.S. Pat. No. 6,127,175, issued to Vigne, et al., discloses a stablytransfected mammalian cell line which expresses a portion of the E4region of adenovirus, preferably orf6/orf6/7. Such a cell line is usefulfor complementation of recombinant Ad genomes deficient in the E4region.

Compositions, including vaccine compositions, comprising the disclosedadenoviral vectors are an important aspect of the present invention.These compositions can be administered to mammalian hosts, preferablyhuman hosts, in either a prophylactic or therapeutic setting. Potentialhosts/vaccines include but are not limited to primates and especiallyhumans and non-human primates, and include any non-human mammal ofcommercial or domestic veterinary importance. Compositions comprisingrecombinant chimpanzee adenoviral vectors may be administered alone orin combination with other viral- or non-viral-based DNA/proteinvaccines. They also may be administered as part of a broader treatmentregimen.

In a particular embodiment of the invention, the disclosed vectors maybe used in an immunization protocol designed to break host tolerance toa self-antigen or a tumor-associated antigen. The identification of anumber of TAA has enabled the development of active vaccinationapproaches for the therapy of cancer. Both cell surface antigens andintracellular antigens that are processed and presented provide usefultargets. Generally speaking, the disclosed method of breaking hosttolerance to a self-antigen comprises: (a) stimulating anantigen-specific response to a self-antigen by administering a firstvaccine composition comprising a first ChAd vector or a plasmid vectorcarrying a nucleotide sequence encoding the self-antigen against whichan antigen-specific immune response is desired, and (b) sustaining andexpanding the immune response of (a) by administering a second vaccinecomposition comprising a recombinant ChAd vector of a different serotypecontaining at least a functional deletion of its genomic E1 gene, and inthe site of the E1 gene, a sequence comprising a promoter capable ofdirecting the expression of DNA encoding the same self-antigen deliveredin the priming step, whereby the host mounts an immune response whichhas the effect of breaking tolerance to the self-antigen.

Accordingly, a skilled artisan can utilize this disclosure to designseveral different immunization protocols that may be suitable for use tobreak host tolerance. For example, it may be possible to utilize aprotocol in which the first, or priming immunization comprises plasmidDNA which encodes a particular self-antigen, such as a TAA, and anysubsequent immunizations comprise a ChAd vector. Plasmid DNA sequencescomprising nucleotide sequences that encode self-antigens, may bedelivered intramuscularly, with or without electrostimulation, in one ormore injections. For example, an immunization protocol based on multiple(e.g., 3 or 4 or 5) intramuscular injections of plasmid DNA encoding aTAA via electroporation followed by one or more intramuscular injectionsof a ChAd vector comprising a transgene encoding the same TAA isencompassed by the general method disclosed and claimed herein.

Alternatively, a suitable protocol to break tolerance could involve oneor more priming immunizations with a ChAd or hAd vector comprising atransgene encoding a self antigen, followed by one or more boostingimmunizations with either the same, or a different ChAd vector that isknow to be non cross-reactive with the vector used for the primingimmunization(s). For example, an immunization protocol using ChAd3 forpriming and ChAd6 for boosting, or ChAd3 for priming followed by ChAd6and ChAd9 for boosting could be used to break host tolerance. Inparticular embodiments, the invention contemplates the use ofself-antigens comprising at least one tumor associated antigen selectedfrom the group consisting of: HER2/neu, CEA, EpCAM, PSA, PSMA,Telomerase, gp100, Melan-A/MART-1, Muc-1, NY-ESO-1, Survivin,Stromelysin 3, Tyrosinase, MAGE3, CML68, CML66, OY-TES-1, SSX-2, SART-1,SART-2, SART-3, NY-CO-58, NY-BR-62, hKLP2, VEGF. In a particularembodiment, the invention provides a method for inducing an immuneresponse (e.g., humoral or cell-mediated) to a tumor-associated antigenwhich is specific for a selected malignancy by delivering a recombinantchimpanzee adenovirus encoding the TAA to a mammal afflicted withcancer. In a preferred embodiment of this aspect of the invention theelicited immune response constitutes an immune response characterized bythe production of antigen-specific CD4+ and CD8+ T cells.

The immunogenic compositions of the invention can be administered tomammalian hosts, preferably human hosts, in either a prophylactic ortherapeutic setting. Potential hosts/vaccines include but are notlimited to primates and especially humans and non-human primates, andinclude any non-human mammal of commercial or domestic veterinaryimportance. Compositions comprising recombinant chimpanzee adenoviralvectors may be administered alone or in combination with other viral- ornon-viral-based DNA/protein vaccines. They also may be administered aspart of a broader treatment regimen. Suitable compositions, for use inthe methods of the invention may comprise the recombinant viral vectorsof the invention in combination with physiologically acceptablecomponents, such as buffer, normal saline or phosphate buffered saline,sucrose, other salts and polysorbate. It does not cause tissueirritation upon intramuscular injection. It is preferably frozen untiluse. Optionally, a vaccine composition of the invention may beformulated to contain other components, such as but not limited to, anadjuvant, a stabilizer, a pH adjusting agent, or a preservative. Suchcomponents are well known to those of skill in the art.

It is envisioned that the recombinant chimpanzee adenoviruses of theinvention will be administered to human or veterinary hosts in an“effective amount,” that is an amount of recombinant virus which iseffective in a chosen route of administration to transduce host cellsand provide sufficient levels of expression of the transgene to invokean immune response which confers a therapeutic benefit or protectiveimmunity to the recipient/vaccine.

The amount of viral particles in the vaccine composition to beintroduced into a vaccine recipient will depend on the strength of thetranscriptional and translational promoters used and on theimmunogenicity of the expressed gene product. In general, animmunologically or prophylactically effective dose of 1×10⁷ to 1×10¹²particles (i.e., 1×10⁷, 2×10⁷, 3×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 5×10⁸or 1×10⁹, 2×10⁹, 3×10⁹, 5×10⁹) and preferably about 1×10¹⁰ to 1×10¹¹particles is administered directly into muscle tissue. Subcutaneousinjection, intradermal introduction, impression through the skin, andother modes of administration such as intraperitoneal, intravenous, orinhalation delivery are also contemplated.

The recombinant chimpanzee adenoviral vectors of the present inventionmay be administered alone, as part of a mixed modality prime/boostvaccination regimen or in a vaccination regimen based on combination ofmultiple injections of different vector serotypes. Typically, a primingdose(s) comprising at least one immunogen is administered to a mammalianhost in need of an effective immune response to a particular pathogen orself-antigen. This dose effectively primes the immune response so that,upon subsequent identification of the antigen(s), the host is capable ofimmediately mounting an enhanced or boosted immune response to theimmunogen. A mixed modality vaccination scheme which utilizedalternative formulations for the priming and boosting can result in anenhanced immune response. Prime-boost administrations typically involvepriming the subject (by viral vector, plasmid, protein, etc.) at leastone time, allowing a predetermined length of time to pass, and thenboosting (by viral vector, plasmid, protein, etc.). Multipleimmunizations, typically 1-4, are usually employed, although more may beused. The length of time between priming and boost may typically varyfrom about four months to a year, albeit other time frames may be usedas one of ordinary skill in the art will appreciate. Multiple injectionof each vector can be administered within approximately a 2 weeks timeframe, before neutralizing immunity becomes evident.

In some embodiments of this invention, a vaccine is given more than oneadministration of adenovirus vaccine vector, and it may be given in aregiment accompanied by the administration of a plasmid vaccine.Suitable plasmid vaccines for use in combination with the vectorsdisclosed herein comprise a plasmid encoding at least one immunogenagainst which a primed or boosted immune response is desired, incombination with a heterologous promoter, which is capable of directingexpression of the nucleic acid sequences encoding the immunogen(s),operably linked to the immunogen coding sequence, and a transcriptionterminator sequence.

For example, a dosing regimen which utilizes multiple injection ofdifferent serotypes of recombinant replication-defective chimpanzeeadenoviral vectors can be used. Alternatively, an individual may begiven a first dose (i.e., a priming dose) of a plasmid vaccine, and asecond dose (i.e., a boosting dose) which comprises areplication-defective recombinant chimpanzee adenoviral vector whichcomprises a coding sequence for the same immunogen that was delivered inthe plasmid vaccine. Alternatively, the individual may be given a firstdose of a human adenovirus vaccine vector encoding at least oneimmunogen, followed by a second dose comprising a replication-defectiverecombinant chimpanzee adenoviral vector disclosed herein, whichcomprises a coding sequence for the same immunogen that was delivered inthe priming dose. In a second alternative embodiment a vaccinecomposition comprising a vector of the invention may be administeredfirst, followed by the administration of a plasmid vaccine. In any ofthese embodiments, an individual may be given multiple doses of the sameimmunogen in either viral vector or plasmid form. There may be apredetermined minimum amount of time separating the administrations.

In addition to a single protein or antigen of interest being deliveredby the recombinant, replication-defective chimpanzee adenovirus vectorsof the present invention, two or more proteins or antigens can bedelivered either via separate vehicles or delivered via the samevehicle. Multiple genes/functional equivalents may be ligated into aproper shuttle plasmid for generation of a adenovirus pre-plasmidcomprising multiple open reading frames. Open reading frames for themultiple genes/functional equivalents can be operatively linked todistinct promoters and transcription termination sequences.

As shown herein, suitable immunization regimens can employ differentadenoviral serotypes. One example of such a protocol would be a primingdose(s) comprising a recombinant adenoviral vector of a first serotype,for example a ChAd3 or ChAd6 followed by a boosting dose comprising arecombinant chimpanzee adenoviral vector of a second serotype. In analternative embodiment, the priming dose can comprise a mixture ofseparate adenoviral vehicles each comprising a gene encoding for adifferent protein/antigen. In such a case, the boosting dose would alsocomprise a mixture of vectors each comprising a gene encoding a separateprotein/antigen, provided that the boosting dose(s) administersrecombinant viral vectors comprising genetic material encoding for thesame or similar set of antigens that were delivered in the primingdose(s). These multiple gene/vector administration modalities canfurther be combined. It is further within the scope of the presentinvention to embark on combined modality regimes which include multiplebut distinct components from a specific antigen.

Use of recombinant vectors derived from chimpanzee adenoviruses that arenot neutralized by preexisting immunity directed against the viralelements of human vector offers an alternative to the use of human Advectors as vaccine carriers. Because adenoviruses are highlyimmunogenicity, adenoviral vectors are particularly good candidates foruse in the context of a vaccine carrier designed to break host toleranceto a self-antigen. Furthermore, the ability to propagate the chimpviruses in human cells, particularly in the Per.C6™ cell line, with anefficiency comparable to human viruses, offers considerable advantagesboth from a regulatory point of view and for the large scale productionof therapeutics or vaccines. Accordingly, the instant invention providesa collection of chimpanzee adenoviral sequences, vectors and plasmidsthat allow the preparation of recombinant virus which may be used, aloneor in combination, as a vaccine carrier for genetic vaccination.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

The following examples illustrate, but do not limit the invention.

Example 1 Isolation, Cloning, Sequencing and Characterization of ChAdsChimpanzee Adenovirus Isolation

Stool specimens were collected in viral transport medium (VTM; MicrotestM4-R Multi-Microbe Transport Medium, Remel Inc.) then frozen or frozendirectly at −70° C. at NIRC (New Iberia Research Center 4401 W. AdmiralDoyle Drive New Iberia, La. 70560). The specimens were kept frozen at<−70° C. until they were processed for inoculation into cell cultures.At that time, the specimens were thawed and then vortexed in excess ofchilled viral transport medium. After the specimens had dissociated intosuspensions, they were centrifuged for 10 min at 1500-1800 rpm. Thesupernatants were filtered through 0.8 and 0.2 μm syringe filters inseries and then the filtered material was inoculated into cell cultures(200-250 μL into shell vials and 250-300 μL into tube cultures). Eachprocessed specimen was inoculated into tube cultures and shell vialcultures seeded with 293 cells or A549 cells.

Control (positive and negative) cultures were prepared each time a setof samples was inoculated. Once all of the shell vials in a set-up hadbeen inoculated, they were centrifuged at room temperature for 60±10 minat 2000 rpm (900×g). The vials were removed from the centrifugeimmediately after the rotor stopped spinning to prevent heat damage inthe cultures. After centrifugation, the inocula were aspirated from theshell vials, using a fresh sterile Pasteur pipette in each vial toprevent cross-contamination. The cultures were washed three times using1.0-mL fresh culture medium for each wash. Fresh medium (1.0 mL) waspipetted into each vial after the third wash and the shell vials wereplaced in an incubator at 35-37° C. for three to four days (approx. 96hr).

At the end of the culture period, the supernatants were aspirated fromthe cultures and the cell layer in each vial was washed twice withImmunofluorescence Assay (IFA) Buffer using approximately 1.0 mL bufferwith each wash. The cells were fixed by adding 1.0 mL refrigeratedacetone to each vial (10 min at 2-8° C. Acetone-cleaned slides werelabeled with the specimen identification number(s) associated with theshell vial coverslips. The shell vial coverslips were processed forfluorescence labeling of Adenovirus-infected cells using a primary mouseanti-adenovirus antibody [MAB8052, CHEMICON®]. The slides are evaluatedwith the aid of a fluorescence microscope. Each preparation was scannedusing the 10× objective noting the extent of immunofluorescence coverageacross the well (1+ to 4+). The presence or absence of specificimmunofluorescence was confirmed using the 40× objective. Tube cultureswere inoculated in the same sequence as described for the shell vials(e.g., negative control first, followed by clinical specimens andpositive controls). The inocula were allowed to adsorb for 60-120 min at36-38° C. After the adsorption period, the specimens/controls wereaspirated from the tubes and replaced by fresh culture medium.

Three to four days post-inoculation, and once a week thereafter, themedia was aspirated from the culture tubes and replaced with 1.5 mLfresh media. Culture tubes were visually monitored for CPE at leastevery other day for at least 21 days after inoculation. Culturesinoculated with chimp specimens were compared against the controls andrated by observing the CPE extent. Cultures showing no CPE were passedto fresh tube cultures after 14 days; culture tubes that were negativefor CPE after 21 days were considered negative. Culture tubes with3-4+CPE were vortexed for 10 seconds. The cells were scraped from thewall of the tube using a sterile 1.0 mL serological pipette andsuspended in the culture supernatant. After labeling a 5 mL snap captube with the specimen identification number and date and stored at −70°C. 500 μL of the cell suspension was transferred from the culture tubeinto the snap cap tube and stored for up to one day at 2-8° C. until itwas processed using an indirect immunofluorescent antibody technique todetect adenovirus (equivalent to procedure for staining shell vials).

Chimpanzee Adenovirus Amplification

Wild type chimp adenoviruses CV32, CV33, CV23 and CV68 purchased fromthe ATCC® (American Type Culture Collection) (ATCC® Accession Numbers:CV32, VR-592; CV-33, VR-593;) or from Esoterix Inc. Austin, Tex. andoriginal isolates were propagated as follows by using the humanE1-expressing cell line PER.C6™ or 293. Briefly, cells were cultivatedin Dulbecco's Modified Eagles Medium (DMEM; GibcoBRL, Life Technologies)supplemented with 10% Fetal Bovine Serum (FBS GibcoBRL, LifeTechnologies), 1% Penicillin-Streptomycin, 2 mM Glutamine and 10 mMMgCl₂ (Per.C6™). Adenovirus infection was carried out in DMEMsupplemented with 5% Horse Serum (GibcoBRL, Life Technologies). Infectedcells and medium were collected when 100% of the cells exhibitedvirus-induced cytopathic effect (CPE) and lysed by three cycles offreezing and thawing.

All wild type chimp adenoviral (CV) stocks were cloned by infecting 293cells seeded in 96-well plates, after the first passage ofamplification. The virus cloning was performed by limiting dilution ofthe cell lysate obtained at the first passage of the virusamplification. Five isolated clones were picked up and seriallypropagated. After 3-4 serial passages of amplification, a large-scalepreparation of adenovirus was performed on cells planted on 5 two-layercell-factories (NUNC) (200 millions of cells/cell factory). Purifiedviral particles were obtained from cell lysate by twoultra-centrifugation steps on cesium chloride density gradients.

Sequencing of Viral Genomic DNA

Genomic DNA was isolated from 3×10¹² pp of purified virus preparation bydigestion with Proteinase K (0.5 mg/ml) in 1% SDS-TEN (2 hrs at 55° C.).After a Phenol-Chloroform extraction and Ethanol precipitation, thegenomic DNA was resuspended in water and submitted for genomicsequencing.

For full length Ad genome sequencing, the purified viral DNA wasnebulized to produce randomly sheared fragments. The DNA fragments wereblunt-ended with the klenow fragment of E. coli DNA polymerase andpolynucleotide kinase. The blunt end fragment were run on a low meltingpoint agarose gel to purify the fragments in the size range of 1-3 kband cloned into the SmaI site of pUC19 vector to create a shotgunlibrary. The ligations were used to transform competent XL1-BLUE® MRF’cells. Positive colonies were identified by white/blue screening on LBagar containing X-gal and IPTG. Three to four 96-well block of plasmidDNA were isolated from the library and sequenced with pUC forward andreverse primers. All sequencing reads were screened for quality andvector sequence using the Phred-Phrap software package. The reads thatpassed the screening were assembled into contigs. Primers were designedto directly sequence the adenoviral DNA for closing the gaps anddetermine the DNA sequence of both ends.

Complete viral genome sequencing was obtained for selected virusesincluding ChAd3 (SEQ ID NO: 1), ChAd6 (SEQ ID NO: 2), CV32 (SEQ ID NO:3), CV33 (SEQ ID NO: 4), and CV23 (SEQ ID NO: 5). Table 1 provides datasummarizing the percentage of identity between the nucleotide sequencesof ChAd3, ChAd6, Pan5 (CV23), Pan6 (CV32), Pan7 (CV33), C1 and C68adenoviral genomes. Alignments were calculated using the ALIGN programas part of the FASTA package version 2 (William R. Penson, University ofVirginia; Myers & Miller, CABIOS1989, 4:11-17).

TABLE 1 Percentage of Nucleotide Sequence Identity Between ChimpanzeeAdenovirus Genomes ChAd3 ChAd6 Pan5 Pan6 Pan7 C1 C68 ChAd3 100 68.1 68.568.2 68.3 64.2 68.0 ChAd6 100 95.5 94.5 95.5 73.6 91.4 Pan5 100 94.996.7 73.9 92.7 Pan6 100 95.1 73.6 91.3 Pan7 100 73.8 93.0 C1 100 74.3C68 100

To characterize the new adenoviral isolates (e.g., ChAd20, ChAd4, ChAd5,ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd22,ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 andChAd82) the nucleotide sequence of the hexon and fiber genes were alsodetermined by primer walking. Fiber gene: SEQ ID NOS: 6-15: (SEQ ID NO:6, ChAd20); (SEQ ID NO: 7, ChAd4); SEQ ID NO: 8, ChAd5); SEQ ID NO: 9,ChAd7); SEQ ID NO: 10, ChAd9); SEQ ID NO: 11, ChAd10); SEQ ID NO: 12,ChAd11); SEQ ID NO: 13, ChAd16) SEQ ID NO: 14, ChAd17), SEQ ID NO: 15,ChAd19), and (SEQ ID NO: 58, ChAd8), (SEQ ID NO: 60, ChAd22), (SEQ IDNO: 62, ChAd24), (SEQ ID NO: 64, ChAd26), (SEQ ID NO: 66, ChAd30), (SEQID NO: 68, ChAd31), (SEQ ID NO: 70, ChAd37), (SEQ ID NO: 72, ChAd38),(SEQ ID NO: 74, ChAd44), (SEQ ID NO: 76, ChAd63) and (SEQ ID NO: 78,ChAd82). FIGS. 20A-20G provide a comparison of the amino acid sequencesof the fiber proteins of the ChAd isolates disclosed and claimed herein.

The hexon gene sequences are set forth in SEQ ID NOS: 16-25: (SEQ ID NO:16, ChAd20); SEQ ID NO: 17, ChAd4); SEQ ID NO: 18, ChAd5); SEQ ID NO:19, ChAd7); SEQ ID NO: 20, ChAd9); SEQ ID NO: 21, ChAd10); SEQ ID NO:22, ChAd11); SEQ ID NO: 23, ChAd16); SEQ ID NO: 24, ChAd17) SEQ ID NO:25, ChAd19), (SEQ ID NO: 97, ChAd8), (SEQ ID NO: 99, ChAd22), (SEQ IDNO:101, ChAd24), (SEQ ID NO: 103, ChAd26), (SEQ ID NO: 105, ChAd30),(SEQ ID NO: 107, ChAd31), (SEQ ID NO: 109, ChAd37), (SEQ ID NO: 111,ChAd38), (SEQ ID NO: 113, ChAd44), (SEQ ID NO: 115, ChAd63) and (SEQ IDNO: 117, ChAd82). FIGS. 31A-31J provide a comparison of the amino acidsequences of the hexon proteins of the ChAd isolates disclosed andclaimed herein.

Chimpanzee Adenovirus Classification

Classification of the different chimp adenoviral strains follows thealready proposed classification of human adenovirus serotypes into 6subgroups (Horowitz, M S (1990) Adenoviridae and their replication. InVirology B. N. Fields and D. M. Knipe, eds (Raven Press, New York) pp.1679-1740) and it was obtained by amino acid and nucleotide sequencealignment by using Align X program (Informax, Inc).

An initial classification of the new isolates was obtained by looking atthe restriction pattern of the viral genome with different restrictionendonucleases and by sequence analysis of the hypervariable region 7(HVR7) of the hexon gene. To this end two primers were designed on thehighly conserved regions flanking HVR7: TGTCCTACCARCTCTTGCTTGA (SEQ IDNO. 45) and GTGGAARGGCACGTAGCG (SEQ ID NO. 46). The HVR7 was amplifiedby PCR using purified viral DNA or crude 293 lysate as template and thensequenced. Based on HVR7 sequence analysis we classified the newisolated viruses into the subgroups (A-F) proposed for human Ad viruses(Horowitz, M S (1990) Adenoviridae and their replication. In Virology B.N. Fields and D. M. Knipe, eds (raven Press, New York) pp. 1679-1740).

The phylogenetic tree presented in FIG. 35 was obtained by alignment ofhuman and chimp adenovirus hexon amino acid sequences. The results areconsistent with the initial classification based on nucleotide sequencealignment limited to hexon HVR7 by using Align X program (Informax,Inc). The tree was deduced from a multiple sequence alignment offull-length hexon peptide sequences using a PAUPSEARCH (WisconsinPackage Version 10.3, Accelrys Inc.) and visualized and manipulated withTREEVIEW. Bootstrap confidence analysis was performed using thePAUPSEARCH program as implemented in the Wisconsin Package. For each ofthe alignments the program was run on 1000 replicates using “HeuristicSearch” as search criterion and Maximum Parsimony as the optimalitycriterion and confidence values reported were taken from a 50%majority-rule consensus.

Example 2 ChAd Shuttle Vector and Expression Vector Construction andRescue Vector Construction and Rescue

Genomic viral DNA was cloned into a standard plasmid vector byhomologous recombination with an appropriate shuttle vector containingviral DNA sequences derived from both left and right end of viral genome(FIG. 2). As described more fully below, the sequence homology observedbetween viruses classified in the same serotype subgroup was exploitedto develop group-specific shuttle vectors. Genomic viral DNA of Chimpadenovirus classified into subgroup D and E resulted to be sufficientlyhomologous to allow the construction of a common shuttle vector in orderto clone viruses belonging to both subgroups.

Construction of a Subgroup D/E Shuttle Vector

The ChAd6 viral genome was fully sequenced (SEQ ID NO: 2) and theinformation obtained was used to construct a shuttle vector tofacilitate cloning by homologous recombination of subgroup D and Echimpanzee adenovirus.

Construction of the ChAd6 shuttle vector, referred to herein as pARSChAd6-3 is described in FIG. 1. FIG. 32 provides a list of theoligonucleotide sequences (SEQ ID NOS: 26-40 and SEQ ID NOS: 45-46) usedin the cloning experiments described herein. Briefly, 457 bp derivingfrom the left end of ChAd6 DNA were amplified by PCR with theoligonucleotides 5′-ATGGAATTCGTTTAAACCATCATCAATAATATACCTC-3 (SEQ ID NO:27) and 5′-CGCTGGCACTCAAGAGTGGCCTC-3′ (SEQ ID NO: 28) digested withEcoRI and SnaBI and cloned into pNEBAd35-2 cut EcoRI-SnaBI, generatingpNEBChAd6-L1. The right ChAd6 ITR (bp 36222 to by 36648) was amplifiedby PCR using the oligonucleotides: 5′-ATGAAGCTTGTTTAAACCCATCATCAATAATATACCT-3′ (SEQ ID NO: 29) and5′-ATCTAGACAGCGTCCATAGCTTACCG-3′(SEQ ID NO: 30) digested withrestriction enzymes HindIII and XbaI and cloned into pNEBChAd6-L1HindIII-XbaI digested thus generating pNEBChAd6-RLI. Finally, the DNAfragment corresponding to nucleotides 3426-3813 of the ChAd6 genomic DNAsequence was amplified with the oligonucleotides: 5′ATGCTACGTAGCGATCGCGTGAGTAGTGTTTGGGGGTGGGTGGG-3′ (SEQ ID NO: 31) and5′-TAGGCGCGCCGCTTCTCCTCGTTCAGGCTGGCG-3′ (SEQ ID NO: 32), digested withSnaBI and AscI then ligated with SnaBI-AscI digested pNEBChAd6-RLI thusgenerating pNEBChAd6-RLIdEl.

To improve the efficiency of recombination and plasmid propagation inDH5a E. coli strain, the 1306 bp fragment containing both left and rightITRs of ChAd6 as well as pIX gene fragment was excised by PmeI digestionfrom pNEBChAd6-RLIdEl and transferred to a different plasmid vectorobtained by PCR amplification with the olinucleotides5′-GATCTAGTTAGTTTAAACGAATTCGGATCTGCGACGCG-3′ (SEQ ID NO: 33) and 5′TTCGATCATGTTTAAACGAAATTAAGAATTCGGATCC-3′ (SEQ ID NO: 34) frompMRKAd5SEAP. This final ligation step generated the ChAd6 shuttle vectorpARSChAd6-3.

Construction of a Subgroup C Shuttle Vector

The ChAd3 viral genome was fully sequenced (SEQ ID NO: 1) and theinformation obtained was used to construct a shuttle vector tofacilitate cloning by homologous recombination of subgroup C chimpanzeeadenovirus.

Briefly, the shuttle vector used to clone subgroup C chimp adenovirus,referred to herein as pChAd3EGFP was constructed as follows: a ChAd3 DNAfragment (nt 3542-4105) containing pIX coding region was amplified byPCR with the oligonucleotides 5′-TATTCTGCGATCGCTGAGGTGGGTGAGTGGGCG-3′(SEQ ID NO: 35) and 5′-TAGGCGCGCCCTTAAACGGCATTTGTGGGAG-3′ (SEQ ID NO:36) digested with SgfI-AscI then cloned into pARSCV32-3 digested withSgfI-AscI, generating pARS-ChAd3D. ChAd3 right end (nt 37320-37441) wasamplified by PCR with oligonucleotides 5′-CGTCTAGAAGACCCGAGTCTTACCAGT-3′(SEQ ID NO: 37) and 5′-CGGGATCCGTTTAAACCATCATCAATAATATACCTTATT-3′ (SEQID NO: 38) digested with XbaI and BamHI then ligated to pARS-ChAd3Drestricted with XbaI and BamHI, generating pARS-ChAd3RD. ChAd3 viral DNAleft end (nt 1-460) was amplified by PCR with oligonucleotides5′-ATGGAATTCGTTTAAACCATCATCAATAATATACCTT-3′ (SEQ ID NO: 39) and5′-ATGACGCGATCGCTGATATCCTATAATAATAAAACGCAGACTTTG-3′, (SEQ ID NO: 40)digested with EcoRI and SgfI then cloned pARS-ChAd3RD digested withEcoRI and SgfI, thus generating pARS-ChAd3RLD. The viral DNA cassettewas also designed to contain restriction enzyme sites (PmeI) located atthe end of both ITR's so that digestion will release viral DNA fromplasmid DNA.

Construction of a Subgroup B Shuttle Vector

The construction of subgroup B shuttle followed the already describedstrategy for subgroup C and D/E shuttle constructions. In brief,pARS-ChAd3RLD was modified by substituting the left end, the pIX region,the right end with the corresponding fragments of ChAd30. In additionthe E4 region of ChAd30 was substituted with Ad5 E4orf6 that was clonedunder the ChAd30 E4 promoter control. The shuttle plasmid wasdenominated pChAd30 EGFP shuttle vector.

Construction of ΔE1 Chimp Adenoviral Vectors

Subgroup B: Subgroup B chimp adenovirus vectors were constructed byhomologous recombination in E. coli strain BJ5183. BJ5183 cells wereco-transformed with pChAd30EGFP shuttle vector digested with BstEII andBst1107I and ChAd8 and ChAd30, purified viral DNA. Homologousrecombination between pIX genes, right ITR DNA sequences present at theends of linearized pChAd30EGFP shuttle and viral genomic DNA allowed itsinsertion in the plasmid vector, deleting at the same time the E1 regionthat was substituted by EGFP expression cassette. Expression cassettesbased on human cytomegalovirus (HCMV) promoter and bovine growth hormonepolyadenylation signal (Bgh polyA) were constructed to express secretedalkaline phosphatase (SEAP), EGFP, HIV gag, HCV NS region (as describedin FIG. 3 for ChAd6 shuttle vectors) as well as tumor-associatedantigens like CEA and HER2/neu from human and Rhesus monkey origin. Allexpression cassette were inserted into ChAd30 vectors by homologousrecombination.

Subgroup C: Subgroup C chimp adenovirus vectors were constructed byhomologous recombination in E. coli strain BJ5183. BJ5183 cells wereco-transformed with pChAd3EGFP shuttle vector digested with BstEII andBst1107I and ChAd3, ChAd11, ChAd19 and ChAd20 purified viral DNA.Homologous recombination between pIX genes, right ITR DNA sequencespresent at the ends of linearized pChAd3EGFP and viral genomic DNAallowed its insertion in the plasmid vector, deleting at the same timethe E1 region that was substituted by EGFP expression cassette.Expression cassettes based on human cytomegalovirus (HCMV) promoter andbovine growth hormone polyadenylation signal (Bgh polyA) wereconstructed to express secreted alkaline phosphatase (SEAP), EGFP, HIVgag, HCV NS region (as described in FIG. 3 for ChAd6 shuttle vectors) aswell as tumor-associated antigens like CEA and HER2/neu from human andRhesus monkey origin.

Subgroups D and E: In order to construct ΔE1 vectors based on subgroup Dand E chimp adenovirus, the shuttle vector pARS ChAd6-3 was digestedwith AscI and co-transformed into E. coli strain BJ5183 with CV32, CV33,CV68, ChAd4, ChAd5, ChAd6, ChAd7, ChAd9, ChAd10 and ChAd16 purifiedviral DNA. Homologous recombination between DNA sequences from pIX genesand right ITR present at the ends of linearized pARS ChAd6-3 and viralgenomic DNA allowed its insertion in the plasmid vector, deleting at thesame time the E1 region (FIGS. 2 and 4).

Expression cassettes based on human cytomegalovirus (HCMV) promoter andbovine growth hormone poly-adenylation signal (Bgh polyA) wereconstructed to express secreted alkaline phosphatase (SEAP), EGFP, HIVgag, HCV NS genes (FIG. 3) as well as tumor-associated antigens like CEAand HER2/neu of human and Rhesus monkey origin. All the expressioncassette were inserted into the single SnaBI site of pARS ChAd6-3 vectorto be transferred by homologous recombination into the ΔE1 adenoviruspre-plasmids as described in FIG. 4.

Rescue and Amplification of ΔE1 Vectors

5×10⁶ PER.C6™ cells planted on 6 cm cell culture dishes were transfectedwith 10 micrograms of cloned viral vector released from plasmidsequences by endonuclease digestion. DNA transfection was performedusing LIPOFECTAMINE® reagent (Invitrogen). Transfected cells and culturemedium were collected 5-10 days post-transfection and lysed byfreeze-thaw. Rescued vectors were then amplified by serial passaging on293 or PER.C6™ cells. A large-scale amplification was performed byinfecting cells planted on 5-10 cell-factories (NUNC, Inc.) on a totalof 1-2×10⁹ cells. A purified vector preparation was obtained on cesiumchloride gradient by two ultra-centrifuge runs, dialyzed against PBScontaining 10% glycerol and stored at −70° C. in aliquots.

Example 3 Neutralization Studies

Neutralization assays were carried out in order to evaluate theprevalence in human sera of neutralizing antibodies against thechimpanzee adenoviruses disclosed herein. The assay evaluated theeffects of serum preincubation on the ability of chimp adenovirusescarrying the gene for secreted alkaline phosphatase (SEAP) to transducehuman 293 cells. The neutralization titer is defined as the dilution ofserum giving a 50% reduction of the SEAP activity observed in thepositive control with the virus alone.

From 2×10⁶ to 1.5×10⁷ physical particles of CV33-SEAP, CV32-SEAP andChAd3-SEAP vector were diluted in 100 μl of complete medium and added toan equal volume of human or chimp serum diluted in complete medium. Eachserum samples was tested at various dilutions (five 4-fold incrementsstarting from 1/18 dilution through 1:4608). Samples were pre-incubatedfor one hour at 37° C. and then added to 293 cells seeded into 96-wellplates (3×10⁴ cells/well). The inoculum was removed after one hour ofincubation, the cells were re-fed with fresh medium and, 24 hours later,50 μl of medium was removed and the SEAP activity was measured by achemiluminescent assay. The neutralization titer is defined as thedilution of serum giving a 50% reduction of the SEAP activity observedin the positive control with the virus alone. A panel of 100 human serawas tested for ChAd neutralization activity. In parallel the same panelwas tested on Ad5 SEAP vector.

TABLE 2 Prevalence of Neutralizing Antibodies Against ChimpanzeeAdenovirus Virus titer hAd5 CV32 CV33 ChAd3 ChAd30 ChAd9 ChAd10 <200 77%96% 100% 92% 100% 92% 100% >200 33%  4%  0%  8%  0%  6%  0%

The result provided in Table 2 indicates that a very low prevalence inhuman sera of neutralizing antibodies directed against vector derivedfrom chimpanzee adenoviruses. Only four sera showed a titer over thethreshold of 200 on CV32 vector while 8 showed a titer over 200 on ChAd3SEAP vector. On the contrary, the panel of chimp sera examined showed avery high prevalence of anti-Chimp Ad immunity. These findings confirmthat as expected, vectors based on chimp Ads have a very little chanceto be neutralized in humans. Therefore they represents an ideal solutionto the problem of the pre-existing anti-human Ad immunity that limitsthe administration of viral vectors based on common human Ad serotypessuch as Ad5.

Example 4 ChAd Vector Tropism

Gene transfer efficacy mediated by Ad5 and ChAd vectors was assessed byEGFP expression on a panel of human primary cells of differenthistological origin. Human chondrocytes, osteoblasts, keratinocytes,melanocytes, skeletal muscle cells and melanocytes were cultivatedaccording to manufacturer indication. Human monocytes, immature andmature dendritic cells (DC) were obtained as described (Romani, N. etal. 1996, J. Immunol. Methods, 196,137.). Transduced, fluorescent cellswere detected by FACS analysis. The panel of human primary cells testedincludes cells that are important target cells for different therapeuticstrategies based on in vivo as well as ex vivo gene transfer in thefield of cardiovascular disease, rheumatoid arthritis, tissueengineering (bone, skin, and cartilage), and vaccination. The resultspresented in FIG. 38A-D suggests that different chimp adenoviruses canrecognize receptors alternative to CAR as demonstrated by thedifferential efficiency of infection of the different cell types.

Murine Immunization Studies

Methods and Materials

Immunization Protocols and Splenocyte/PBMC Preparation

Immunizations: Mice were immunized with the selected adenovirusesdiluted in 0.1 ml of buffer. Each vector dose was divided in two aliquotof 50 μl and injected in both quadriceps of mice.

Splenocyte Preparation Mice were sacrificed 3 weeks post-injection andtheir spleens excised and transferred in 10 ml of R10 (10% FCS, 55 mM2-mercaptoethanol, 1M HEPES buffer, 2 mM L-glutamine, 1×penicillin-streptomicine solution in RPMI medium 1640). Spleens wereminced through a steel screen and, after the screen was washed with 2 mlof R10, splenocytes were transferred in a 50 ml FALCON™ tube andcentrifuged at 1200 rpm, 10 min, room temperature (rt). Supernatant wasremoved and 3 ml of ACK lysis buffer (Gibco BRL Formulation#79-0422DG)were added. Cells were incubated 5 min, rt. 45 ml of 1×PBS were addedand tubes were centrifuged as above. After washing with 30 ml of R10,cells were resuspended in 5 ml of R10, filtered through a 70 m Nyloncell strainer (FALCON™ 2350). 10 μl of cells were diluted with 990 μlTurk's solution (Merck 040417345) and counted. Cells were finallydiluted to 10⁷ cells/ml in R10.

Peripheral blood mononuclear cell (PBMC) preparation: Mice blood samples(150 ul) were transferred to 2 ml eppendorf tubes with 50 ul PBS/2%EDTA. 1 ml ACK buffer was added to each tube. Gently mixed and incubatedat RT for 5 min. Samples were centrifuged at 1500 rpm in microcentrifugefor 5 min. Supernatant was discharged white cell pellets deriving fromthe same immunized cohorts were combined. ACK buffer incubation wasrepeated then pellets of PBMC were resuspended in 1 ml of R10 medium.

IFN-γ ELISPOT Assay

MILLIPORE® MAIP 45 plates were coated with 100 μl/well of purified ratanti-mouse IFN-γ monoclonal antibody (PHARMINGEN™, cat. 551216) dilutedat 2.5 μg/ml in PBS and incubated over-night (o/n) at 4° C. Plates werewashed 2× with sterile PBS and un-specific binding sites were blocked byincubation for 2 hrs in the CO₂ incubator with 200 μl/well of R10. Inthe immunization experiments with Ad vectors expressing HIV gag, a 9-merpeptide (AMQMLKETI, a CD8 HIV gag epitope mapped in Balb/C mice) (SEQ IDNO: 47) was diluted to 2 μg/ml in R10 and added to the wells in theamount of 50 μl/well. In immunization experiments conducted with HCV-NSexpressing vectors, a pool of peptides covering NS3 helicase domain aswell a 9-mer peptide representing a mapped CD8 epitope comprised inhelicase domain were used. Immunization experiments with ChAdsexpressing human CEA antigen were evaluated by pools of overlapping15-mer peptides covering the entire amino acid sequence. As controlsDMSO and Concanavalin A were used. Cells were added to each well at theamount of 5×10⁵ and 2.5×10⁵. After an o/n incubation in the CO₂incubator, plates were washed with 0.05% TWEEN® 20 (Polysorbate 20)/PBSand 50 μl/well of biotinylated rat anti-mouse IFN-γ monoclonal antibody(PharMingen cat. 554410) diluted 1/250 in assay buffer (5% FBS, 0.005%TWEEN® 20 (Polysorbate 20), PBS) were added. Plates were incubated o/nat 4° C. and washed as above. Streptavidin-alkaline phosphataseconjugate (BD554065) was diluted 1/2500 in assay buffer and added in theamount of 50 μl/well for 2 hrs rt. After washing, plates were developedadding 50 μl/well of BCIP/NBT 1-STEP™ solution (Pierce 34042). Reactionwas stopped by washing wells with deionized water. Spots wereautomatically counted by an ELISPOT reader.

Murine IFN-γ Intracellular Staining (ICS)

Splenocytes were diluted at 2×10⁶ cells in 1 ml of R10 and stimulatedwith the same antigens described above at the concentration of 2 μg/ml.As controls, DMSO and Staphylococcal Enterotoxin B (SEB) were used.After an overnight incubation in the CO₂ incubator, cells were washedwith FACS buffer (1% FCS, 0.01% NaN3, PBS) and purified anti-mouseCD16/CD32 Fc block (clone 2.4G2, PHARMINGEN™ cat. 553142) was diluted1/25, added in the amount of 100 μl/sample and incubated for 15 min at4° C. Cells were washed in FACS buffer and APC conjugated anti-mouseCD3e (clone 145-2C11, PHARMINGEN™ #553066), PE conjugated anti-mouse CD4(clone L3T4, BD PHARMINGEN™ cat. 553142) and PerCP conjugated anti-mouseCD8a (clone 53-6.7, PHARMINGEN™ cat. 553036) diluted 1:50 in FACS bufferwere added in the amount of 100 μl/sample. Cells were incubated 30 minrt, washed, fixed and permeabilized (Becton Dickinson, FACS Perm 2) andincubated with FITC conjugated anti-mouse IFN-γ-PHARMINGEN™ cat. 554411)diluted 1:50 in PermWash (100 ul/sample) for 30 min at RT. After washingcells were resuspended in 500 ul 1% formaldehyde/PBS and intracellularcytokine staining (ICS) analyzed on a FACSCALIBUR™ flow cytometer, usingCELLQUEST™ software (Becton Dickinson).

Example 5 ChAd Vectors Elicit Strong CMI Responses in Mice

The ability of the ChAd vectors disclosed herein to elicit acell-mediated immune response (CMI) was evaluated in mice using vectorsexpressing an HIV gag transgene. Briefly, groups of 5 Balb/C mice wereinjected with ten-fold increasing doses of the different vectorsstarting from 10⁵ up to 10¹⁰ vp/mouse.

The strength of the immune response was determined three weeks after theinjection by quantifying gag-specific CD8+ T cells in the splenocytes.The number of IFN-γ secreting CD8+ T cells was determined by ELISPOTassay or by IFN-γ intracellular staining and FACS analysis afterstimulation in vitro with a peptide reproducing a gag CD8+ T cellepitope mapped in Balb/C mice.

The results obtained from the 5 immunized animals, reported in Table 3,are expressed as spot forming cells per 10⁶ splenocytes. Shown are thenumber of spot forming cells per million splenocytes followingincubation with 9-mer CD8+ gag epitope or with gag peptide pool. The gagpeptide pool consisted of 20-aa peptide overlapping by 10aa encompassingthe entire gag sequence. Positive values are reported in bold.

The data provided in Table 3 indicate that the administration of theChAd vectors disclosed and claimed herein elicits a strong cell mediatedimmune response which is comparable to the response elicited by hAd5. Bylooking at the lowest vector dose resulting in a positive immunizationresult (immunization breakpoint), we ranked the potency of the differentvectors being subgroup C ChAd3gag the most potent with a breakpoint at10⁶ pp vector dose. Ranking by immunization break-points is shown inFIG. 33.

TABLE 3 Gag-Specific T Cell Response in Balbc Mice Immunized withChimpanzee Ad Vectors 10{circumflex over ( )}5 vp 10{circumflex over( )}6 vp 10{circumflex over ( )}7 vp 10{circumflex over ( )}8 vp10{circumflex over ( )}9 vp 10{circumflex over ( )}10 vp Vaccinationmock Gag mock Gag mock Gag mock Gag mock Gag mock Gag ChAd3DE1gag 1 neg1 944 1 1298 1 1258 NT NT NT NT 3 neg 1 1039 1 1958 1 1962 NT NT NT NT 1neg 1 859 1 1923 1 1931 NT NT NT NT 1 neg 1 1620 1 1386 1 1369 NT NT NTNT 1 neg 1 1529 5 1442 4 1436 NT NT NT NT CV33DE1gag NT NT 1 neg 2 475 12910 NT NT NT NT NT NT 1 neg 1 433 1 401 NT NT NT NT NT NT 1 neg 1 243 1634 NT NT NT NT NT NT 1 neg 1 505 2 3457 NT NT NT NT NT NT 1 neg 1 683 21684 NT NT NT NT CV68DE1gag NT NT 3 neg 1 340 2 332 0 406 2 635 NT NT 1neg 1 512 0 536 1 256 3 1172  NT NT 0 neg 2 458 3 944 2 462 2 505 NT NT7 neg 0 148 1 519 0 488 2 1184  NT NT 0 neg 2 1418 1 243 0 240 1 789ChAd9DE1gag NT NT 1 neg 7 369 1 609 NT NT NT NT NT NT 1 neg 1 508 1 739NT NT NT NT NT NT 1 neg 1 299 16 291 NT NT NT NT NT NT 1 neg 2 507 8 926NT NT NT NT NT NT 0.5 neg 1 36 40 1034 NT NT NT NT ChAd10DE1gag NT NT 1neg 1 83 1 822.5 NT NT NT NT NT NT 1 neg 1 42.5 1 1033 NT NT NT NT NT NT1 neg 1 48 1 1339.5 NT NT NT NT NT NT 1 neg 4 51 1 1132 NT NT NT NT NTNT 1 neg 1 466.5 1 521.5 NT NT NT NT ChAd6DE1gag NT NT 1 neg 1 34 1 721NT NT NT NT NT NT 1 neg 10 4 1 560 NT NT NT NT NT NT 1 neg 1 24 1 624 NTNT NT NT NT NT 1 neg 1 225 3 3002 NT NT NT NT NT NT 1 neg 1 276 4 1738NT NT NT NT ChAd11DE1gag 1 neg 1 neg 0 573 NT NT NT NT NT NT 0 neg 0 neg0 919 NT NT NT NT NT NT 0 neg 1 neg 1 1438 NT NT NT NT NT NT 2 neg 0 neg0 0 NT NT NT NT NT NT 1 neg 1 neg 0 456 NT NT NT NT NT NT ChAd20DE1gag 0neg 0 neg 0 1 NT NT NT NT NT NT 2 neg 0 neg 0 408 NT NT NT NT NT NT 0neg 0 neg 0 414 NT NT NT NT NT NT 1 neg 0 neg 0 2 NT NT NT NT NT NT 0neg 0 neg 1 311 NT NT NT NT NT NT ChAd7DE1gag NT NT 1 neg 1 neg 1 1044NT NT NT NT NT NT 3 neg 1 neg 1 606 NT NT NT NT NT NT 1 neg 8 neg 1 407NT NT NT NT NT NT 1 neg 1 neg 2 567 NT NT NT NT NT NT 1 neg 3 neg 1 1677NT NT NT NT CV32DE1gag NT NT NT NT 1 neg 0 83 0 291 0 194 NT NT NT NT 3neg 0 382 0 805 2 380 NT NT NT NT 0 neg 1 97 0 136 1 501 NT NT NT NT 1neg 5 96 4 1162  0 1115  NT NT NT NT 2 neg 1 328 NT NT 0 596 ChAd4DE1gagNT NT 1 neg 0 neg 0 0 NT NT NT NT NT NT 0 neg 0 neg 0 159 NT NT NT NT NTNT 0 neg 0 neg 0 1 NT NT NT NT NT NT 1 neg 0 neg 0 234 NT NT NT NT NT NT1 neg 0 neg 1 0 NT NT NT NT ChAd16DE1gag NT NT 0 neg 0 neg 0 243 NT NTNT NT NT NT 0 neg 0 neg 1 296 NT NT NT NT NT NT 0 neg 2 neg 1 68 NT NTNT NT NT NT 0 neg 0 neg 1 433 NT NT NT NT NT NT 1 neg 0 neg 1 28 NT NTNT NT

Example 6 ChAd3 and CV33 GAG Vectors Elicit a CMI Response Characterizedby GAG-Specific CD8+ T Cells

In order to characterize the CMI response elicited in response to theChAd vectors comprising HIV gag transgene, splenocytes pooled fromcohorts of five mice immunized with different doses of vector wereanalyzed by intracellular IFN-γ staining. The data shown in table 3 andtable 4 were collected in separate experiments.

Splenocytes were diluted at 2×10⁶ cells in 1 ml of R10 and stimulatedwith the same antigens described above at the concentration of 2 μg/ml.As controls, DMSO and SEB (Staphylococcal Enterotoxin B) were used.After an o/n incubation in the CO₂ incubator, cells were washed withFACS buffer (1% FCS, 0.01% NaN₃, PBS) and purified anti-mouse CD16/CD32Fc block (clone 2.4G2, PHARMINGEN™ cat. 553142) was diluted 1/25, addedin the amount of 100 μl/sample and incubated for 15 min at 4° C. Cellswere washed in FACS buffer and APC conjugated anti-mouse CD3e (clone145-2C11, PHARMINGEN™ #553066), PE conjugated anti-mouse CD4 (cloneL3T4, BD PHARMINGEN™ cat. 553142) and PerCP conjugated anti-mouse CD8a(clone 53-6.7, PHARMINGEN™ cat. 553036) diluted 1:50 in FACS buffer wereadded in the amount of 100 μl/sample. Cells were incubated 30 min rt,washed, fixed and permeabilized (Becton Dickinson, FACS Perm 2) andincubated with FITC conjugated anti-mouse IFN-γ-PHARMINGEN™ cat. 554411)diluted 1:50 in PermWash (100 ul/sample) for 30 min at RT. After washingcells were resuspended in 500 ul 1% formaldehyde/PBS and analyzed on aFACSCALIBUR™ flow cytometer, using CELLQUEST™ software (BectonDickinson).

Table 4 provides data summarizing the percentage of gag-specific CD3+ Tcells that were either gag-specific CD8+ or CD4+ T cells. Positiveresults are reported in bold. The data provided herein indicate that thecellular profile of the immune response elicited by ChAd vectors derivedfrom viruses classified into different serotype subgroups (i.e.,subgroups C, D and E) are similar and all of the gag-specific responsescharacterized predominantly by CD8+ T cells. In addition, it is notedthat at high vector doses a gag-specific CD4+ response becomes evidentin all immunization experiments. The ICS assay confirmed that ChAd3vector can stimulate anti-gag CD8+ response at 10⁶ vector dose.

TABLE 4 Characterization of Gag-Specific T Cells in Mice Immunized withChimp Adenovirus Vectors of Different Subgroups 10⁵ 10⁶ 10⁷ 10⁸ 10⁹vaccine DMSO gag DMSO gag DMSO gag DMSO gag DMSO gag ChAd3DE1gag %CD8⁺CD3⁺ NT NT 0.01% 4.65% 0.01% 17.15% 0.04% 24.71% NT NT % CD4⁺CD3⁺ NTNT 0.00% 0.07% 0.03% 0.08% 0.04% 0.28% NT NT CV33DE1gag % CD8⁺CD3⁺ NT NT0.02% 0.01% 0.01% 0.83% 0.03% 8.69% NT NT % CD4⁺CD3⁺ NT NT 0.00% 0.00%0.00% 0.04% 0.01% 0.10% NT NT ChAd9DE1gag % CD8⁺CD3⁺ NT NT 0.02% 0.01%0.01% 0.68% NT NT 0.04% 4.73% % CD4⁺CD3⁺ NT NT 0.00% 0.00% 0.00% 0.00%NT NT 0.00% 0.01% ChAd10DE1gag % CD8⁺CD3⁺ NT NT 0.02% 0.01% 0.01% 0.57%NT NT 0.02% 5.04% % CD4⁺CD3⁺ NT NT 0.00% 0.00% 0.00% 0.00% NT NT 0.00%0.01% ChAd6DE1gag % CD8⁺CD3⁺ NT NT 0.00% 0.01% 0.00% 0.59% 0.01% 14.28%NT NT % CD4⁺CD3⁺ NT NT 0.00% 0.00% 0.00% 0.05% 0.01% 0.12% NT NTChAd7DE1gag % CD8⁺CD3⁺ NT NT 0.01% 0.02% 0.01% 0.00% 0.02% 5.00% NT NT %CD4⁺CD3⁺ NT NT 0.00% 0.01% 0.00% 0.00% 0.01% 0.21% NT NT

Example 7 ChAd Vectors Elicit HCV NS-Specific T Cell Response

The potency of CV32-NSmut and CV33-NSmut vectors was evaluated inC57/Black6 mice relative to the potency of MRKAd6NSmut. The animals wereinjected with 10-fold increasing doses of vector starting from 10⁷ up to10⁹ vp/mouse. CMI was analyzed 3 weeks after a single injection by IFN-γELISPOT and IFN-γ intracellular staining by stimulating T cells with a9-mer peptide reproducing a CD8+ T cell epitope mapped in the helicasedomain of NS3 protein. The data provided in Table 5 summarize the numberof spot-forming cells per million splenocytes following incubation inabsence (mock) or in presence of NS3 9-mer peptide.

The data indicate that both CV32 and CV33 vectors expressing HCV-NSstimulate strong T cell responses. Based on the observation that thefirst positive result for the CV32 vector was obtained by injecting 10⁹vp/dose, the immunization potency of CV32DE1E3 NSmut vector appears tobe approximately 100-fold lower than human subgroup C Ad6DE1E3 NSmutvector. The parallel experiment with MRKAd6NSmut indicated that a doseof 10⁷ vp/animal was sufficient to stimulate cell mediated immunity.Therefore, these results confirm the lower immunization potency ofCV32-derived vectors relative to human subgroup C vectors (such as hAd5and hAd6) that was also observed in the experiment with gag expressingvectors (see Table 3).

TABLE 5 HCV NS-Specific T Cell Response in Mice Immunized with Mrkad6Nsmut, CV32NSmut or CV33NSmut 10{circumflex over ( )}7 vp 10{circumflexover ( )}8 vp 10{circumflex over ( )}9 vp 10{circumflex over ( )}10 vpVaccination Mock NS3 mock NS3 mock NS3 mock NS3 MRKAd6NSmut 1 345 1 449NT NT NT NT 1 248 1 1590 NT NT NT NT 1 1 1 549 NT NT NT NT 1 262 NT NTNT NT NT NT NT NT CV33NSmut 1 1 1 195 2 338 NT NT 1 2 1 409 1 1136 NT NT1 1 1 396 1 497 NT NT 1 2 2 172 1 344 NT NT 1 237 1 163 NT NT CV32NSmutneg neg 1 181 1 118 1 176 neg neg 1 71 1 239 1 238 neg neg 1 56 1 862 1555 neg neg 1 459 1 219 1 545 neg neg 1 195 1 123 1 578

Example 8 Anti-Ad5 Pre-Existing Immunity Does Not Abrogate Anti-GAG CMIElicited by ChAd3gag

To evaluate the impact on ChAd3 immunization of the pre-existingimmunity against the high seroprevalent Ad5, 4 cohorts of 5 BalbC micewere pre-immunized with two injection of 10¹⁰ vp of Ad5 wt in thequadriceps at week 0 and 2. As control, 2 cohorts of 5 mice wereinjected at the same time points with buffer only. Cohorts of Ad5pre-immunized mice were then immunized with 10⁶ and 10⁷ vp/mouse ofeither Ad5gag or ChAd3gag vectors. Cohorts of control (naïve) mice wereimmunized with 106 vp/mouse of Ad5gag or ChAd3gag vectors.

Anti-Ad5 and ChAd3 neutralizing immunity was evaluated at week 4 by theneutralization assay described above using Ad5 and ChAd3 SEAP vectors.Anti-gag immunity was evaluated by ELISPOT analysis on purifiedsplenocytes stimulated with gag 9-mer peptide containing a gag epitopemapped in BalbC mice. The results reported in FIG. 36 demonstrated thatAnti-Ad5 immunity does not abrogate anti-gag CMI elicited by ChAd3gagwhile, as expected, anti-Ad5 immunity completely block Ad5gagimmunization.

Example 9 ChAd3hCEA Immunization Elicits a Strong CEA-Specific ImmuneResponse in Transgenic Mice Expressing Human CEA

The ability of the ChAd vectors disclosed and claimed herein to elicitan immune response against a self-antigen therefore breaking thetolerance was also evaluated in transgenic mice expressing human CEA(Clarke, P et al. Cancer Res. (1998) 58(7):1469-77.)

Cohorts of 8 mice were injected in the quadriceps with 10^10 vp ofChAd3hCEA or Ad5hCEA as already described. The immune response againstCEA was followed weekly up to day 75 on PBMC stimulated with a pool of15-mer peptides encompassing human CEA amino acid sequence from aa 497to the end (aa 703). Anti-CEA immunity was evaluated by ICS determiningCD4-CD8+ T cells secreting interferon-γ in response to CEA peptide poolincubation.

The results reported in FIG. 37 demonstrate that ChAd3hCEA vectorimmunization stimulate a more sustained CD8+ T cell response againsthuman CEA than Ad5 expressing the same transgene.

Primate Immunization Studies

Methods and Materials

Immunization Protocol

The ability of the ChAd vectors disclosed and claimed herein to elicitCMI in Rhesus macaques (referred to herein as monkeys) was alsoevaluated. The macaques were anesthetized (ketamine/xylazine) and thevaccines were delivered i.m. in 0.5-mL aliquots into both deltoidmuscles using tuberculin syringes (Becton-Dickinson). In all cases themacaques were between 3-10 kg in weight, and the total dose of eachvaccine was administered in 1 mL of buffer.

Sera and peripheral blood mononuclear cells (PBMC) were prepared fromblood samples collected at several time points during the immunizationregimen. All animal care and treatment were in accordance with standardsapproved by the Institutional Animal Care and Use Committee according tothe principles set forth in the Guide for Care and Use of LaboratoryAnimals, Institute of Laboratory Animal Resources, National ResearchCouncil.

ELISPOT Assay

The IFN-γ ELISPOT assays for rhesus macaques were conducted following apreviously described protocol (Allen et al., 2001 J. Virol.75(2):738-749), with some modifications. For gag-specific stimulation, apeptide pool was prepared from 20-aa peptides that encompass the entireHIV-1 gag sequence with 10-aa overlaps (Synpep Corp., Dublin, Calif.).For HCV NS-specific stimulation 6 peptide pools were prepared from 15-aapeptides that encompass the entire HCV-NS sequence from NS3 to NS5b with10-aa overlaps. HER2/neu and CEA-specific stimulations were performedwith 15-aa peptides that encompass the entire protein sequence with10-aa overlaps.

To each well, 50 μL of 2−4×10⁵ peripheral blood mononuclear cells(PBMCs) were added; the cells were counted using Beckman Coulter Z2particle analyzer with a lower size cut-off set at 80 fL. Either 50 μLof media or the gag peptide pool at 8 μg/mL concentration per peptidewas added to the PBMC. The samples were incubated at 37° C., 5% CO₂ for20-24 hrs. Spots were developed accordingly and the plates wereprocessed using custom-built imager and automatic counting subroutinebased on the IMAGEPRO™ platform (Silver Spring, Md.); the counts werenormalized to 10⁶ cell input.

Intracellular Cytokine Staining (ICS)

To 1 ml of 2×10⁶ PBMC/mL in complete RPMI media (in 17×100 mm roundbottom polypropylene tubes (Sarstedt, Newton, N.C.)), anti-hCD28 (cloneL293, Becton-Dickinson) and anti-hCD49d (clone L25, Becton-Dickinson)monoclonal antibodies were added to a final concentration of 1 μg/mL.For gag-specific stimulation, 10 μL of the peptide pool (at 0.4 mg/mLper peptide) were added. Similar conditions were used for HCVNS-specific stimulation. The tubes were incubated at 37° C. for 1 hr.,after which 20 μL of 5 mg/mL of brefeldin A (Sigma) were added. Thecells were incubated for 16 hr at 37° C., 5% CO₂, 90% humidity. 4 mLcold PBS/2% FBS were added to each tube and the cells were pelleted for10 min at 1200 rpm. The cells were re-suspended in PBS/2% FBS andstained (30 min, 4° C.) for surface markers using severalfluorescent-tagged mAbs: 20 μL per tube anti-hCD3-APC, clone FN-18(Biosource); 20 μL anti-hCD8-PerCP, clone SK1 (Becton Dickinson,Franklin Lakes, N.J.); and 20 μL anti-hCD4-PE, clone SK3 (BectonDickinson). Sample handling from this stage was conducted in the dark.The cells were washed and incubated in 750 μL 1×FACS Perm buffer (BectonDickinson) for 10 min at room temperature. The cells were pelleted andre-suspended in PBS/2% FBS and 0.1 μg of FITC-anti-hIFN-γ, clone MD-1(Biosource) was added. After 30 min incubation, the cells were washedand re-suspended in PBS. Samples were analyzed using all four colorchannels of the Becton Dickinson FACSCALIBUR™ instrument. To analyze thedata, the low side- and forward-scatter lymphocyte population wasinitially gated; a common fluorescence cut-off for cytokine-positiveevents was used for both CD4′ and CD8′ populations, and for both mockand gag-peptide reaction tubes of a sample.

Example 10 A Homologous Prime-Boost Regimen Using ChAd ΔE1-Gag VectorsElicits Gag-Specific T Cells in Monkeys

Cohorts of 3 animals were given intramuscular injection at week 0 andweek 4 of either of the following constructs: 10^ 10 vp of CV-32ΔE1-gag;or 10^10 vp CV33ΔE1-gag; or 10^ 10 vp and 10^8 vp MRKAd5ΔE1gag. PBMCscollected at regular 4-wks intervals were analyzed in an ELISPOT assay.The results provided in Table 6, which indicate the number ofspot-forming cells per million PBMC following incubation in absence(mock) or presence of Gag peptide pool establish that both CV32ΔE1-gagand CV-33ΔE1gag are able to induce significant levels of gag-specific Tcells in non-human primates. It is interesting to note that after asingle dose (wk 4), the CV32ΔE1-gag responses were comparable to MRKAd5μl-gag 10^8 vp dose and lower than that of MRKAd5-gag 10^10 vp/dose.CV33ΔE1-gag 10^10 vp/dose induces a response comparable to that ofMRKAd5-gag 10^10 vp/dose. This result was confirmed at week 8 after thesecond dose.

TABLE 6 Gag-Specific T Cell Response in Monkeys Immunized with Mrkad5ΔE1-Gag, CV32ΔE1-Gag, CV33ΔE1-Gag Vaccination Pre-bleed T = 4 T = 8 T =0 vector dose Monk # Mock Gag Mock Gag Mock Gag CV32ΔE1gag 10{circumflexover ( )}10 vp 01C023 1 0 14 353 3 278 01C029 1 3 13 605 3 419 01C032 10 5 274 1 179 CV33ΔE1gag 10{circumflex over ( )}10 vp 01C033 0 0 9 15451 659 01C036 4 5 4 1540 13 881 01D303 0 3 19 949 10 628 MRKAd5gag10{circumflex over ( )}8 vp 01D267 0 0 4 473 0 341 01D279 1 4 44 831 6336 01D284 4 5 4 264 5 129 MRKAd5gag 10{circumflex over ( )}10 vp 99C2180 3 5 2500 0 1580 99C227 6 1 4 529 5 365 99D185 ND ND 0 425 0 310

Example 11 ChAd Vectors Elicit a HCV NS-Specific T-Cell Response in aHeterologous Prime-Boost Regimen

In a separate experiment, groups of two and three monkeys were givenimmunization at week 0, 4 of MRK Ad6NSoptmut vector at 10^8 or 10^10 vpper animal. The animals were boosted with the same virus at the samedose at week 24 and then boosted again at week 104 with CV33-NSmut at10^10 vp per animal. The results are presented in Tables 7 and 8 whichsummarize the number of spot-forming cells per million PBMC followingincubation in absence (mock) or presence of HCV NS peptide pool.

T cell immunity, as assessed by IFN-γ ELISPOT, showed a peak response atweek 4 after the first dose in the animals injected with 10^10 vp (Table8) and at week 8 (post-dose 2) in the animals injected at 10^8 (Table7). The response was not boosted by the injection at week 24(“homologous boost”), while a strong boost effect was observed after theinjection with CV33-NSmut (“heterologous boost”).

TABLE 7 HCV NS-Specific T Cell Response in Monkeys Immunized with MRKAd6NSoptmut At 10{circumflex over ( )}8 Vp/Animal and Boosted withCV33-Nsmut Vaccine MRKAd6NSoptmut 10{circumflex over ( )}8 vp CV33-NSmut10{circumflex over ( )}10 vp time post-priming I post-priming IIpre-homologous post-homologous pre-heterologous post-heterologous pointdose T = 4 dose T = 8 boost T = 24 boost T = 28 boost T = 104 boost T =108 monkey 95116 138T 95116 138T 95116 138T 95116 138T 95116 138T 95116138T poolF 44 112 77 124 115 176 105 55 120 150 188 2228 poolG 20 211086 1975 201 1105 94 884 120 192 96 4590 poolH 12 18 54 22 169 221 28 981 33 447 543 poolI 14 53 62 47 163 189 96 18 80 67 71 515 poolL 33 8658 44 353 608 235 33 110 131 224 308 poolM 184 75 168 138 204 336 67 4455 46 2028 1570 DMSO 14 3 44 7 104 79 33 6 57 40 33 65

TABLE 8 HCV NS-Specific T Cell Response In Monkeys Immunized MRKAd6NSoptmut At And 10{circumflex over ( )}10 vp/Animal And Boosted WithCV33-Nsmut Vaccine MRKAd6NSoptmut 10{circumflex over ( )}10 vp timepost-priming I post-priming II pre-homologous post-homologous point doseT = 4 dose T = 8 boost T = 24 boost T = 28 monkey 98D209 106Q 113Q98D209 106Q 113Q 98D209 106Q 113Q 98D209 106Q 113Q poolF 3110 263 4041340 300 723 678 61 583 321 123 1438 poolG 2115 642 1008 1070 316 2205685 71 701 251 178 1758 poolH 373 72 19 358 43 43 424 24 42 51 23 18poolI 103 37 347 80 36 531 237 39 169 12 35 485 poolL 149 22 10 93 36 29279 46 48 11 49 51 poolM 314 428 19 153 243 20 333 81 38 38 134 11 DMSO0 1 3 16 16 5 128 8 9 8 10 16 Vaccine CV33-NSmut 10{circumflex over( )}10 time pre-heterologous post-heterologous point boost T = 104 boostT = 108 monkey 98D209 106Q 113Q 98D209 106Q 113Q poolF 204 192 326 15811525 1714 poolG 166 106 625 1118 524 4238 poolH 92 45 55 413 58 211poolI 66 79 376 459 85 2738 poolL 89 109 73 199 76 431 poolM 41 81 9 2281440 227 DMSO 20 51 12 18 13 5

The efficiency of heterologous boost with chimp Ad vectors was evaluatedin a second experiment. Cohorts of three monkeys were immunized at week0 and week 4 with MRKAd5gag (10^10 vp/animal), MRKAd6NSmut (10^10vp/animal) or with the combination of both vectors (10^10 vp/animal eachvector) then boosted with the same immunogen at week 24 (homologousboost).

Homologous boost was performed with the same immunogens; heterologousboost was performed with CV33gag, CV32 NSmut or with the two vectors incombination. The results provided in Table 9 summarize the number ofspot-forming cells per million PBMC following incubation in absence(mock) or presence of HCV NS peptide pool.

The same cohorts were boosted again at week 51 with CV33gag (10^10vp/animal), CV32NSmut (10^10 vp/animal) and with the combination of thetwo vectors (10^10 vp/animal each vector). The results provided in Table9 further indicate that the homologous boost was not efficient since theresponses are below the peak observed at week 4 after the injection ofthe first dose of vaccine. A strong boosting effect was measured byIFN-γ ELISPOT at week 54 after immunization with heterologous chimpvectors.

TABLE 9 Immunization with Chimp Ad vectors efficiently boost Gag and HCVNS-specific T cell response in monkeys immunized with MRK Ad5gag or MRKAd6NSoptmut at 10{circumflex over ( )}10 vp/animal Vaccine MRKAd5gagtime post-dose 1 post-dose 2 pre-homol. point (T = 4) (T = 8) boost (T =24) animal ID 00D105 00D076 00D299 00D105 00D076 00D299 00D105 00D07600D299 poolF 18 35 60 16 29 14 37 76 40 poolG 16 23 49 4 28 31 54 95 106poolH 45 51 57 18 31 42 55 88 55 poolI 21 21 48 4 26 11 19 54 26 poolL15 21 58 9 31 20 71 183 128 poolM 39 24 49 26 14 49 38 93 39 Gag 17642208 2762 574 1906 1959 391 935 702 DMSO 9 13 37 7 14 13 16 76 33Vaccine MRKAd5gag CV33gag time post-homol. pre-heterol. post-heterol.point boost (T = 28) boost (T = 51) boost (T = 54) animal ID 00D10500D076 00D299 00D105 00D076 00D299 00D105 00D076 00D299 poolF 37 8 14 3727 44 43 44 70 poolG 81 2 46 36 27 37 84 108 109 poolH 47 11 32 69 36 6085 58 120 poolI 38 6 6 22 11 32 33 26 24 poolL 106 6 27 61 21 65 28 4544 poolM 59 6 19 62 23 38 27 19 14 Gag 2123 336 736 485 833 1384 40034333 3863 DMSO 26 3 11 28 19 39 23 16 53 Vaccine MRK Ad5gag +MRKAd6NSmut time post-dose 1 post-dose 2 pre-homol. point (T = 4) (T =8) boost (T = 24) animal ID 00D088 00D099 00D240 00D088 00D099 00D24000D088 00D099 00D240 poolF 438 118 105 720 116 154 206 108 242 poolG 21784 1483 44 362 940 19 234 548 poolH 24 53 8 46 27 19 13 66 93 poolI 8328 9 90 24 8 16 40 68 poolL 13 14 13 16 17 9 28 101 140 poolM 39 31 6101 27 16 21 73 107 Gag 2138 1044 1063 2260 505 819 454 241 456 DMSO 5 63 8 5 1 10 18 43 Vaccine MRK Ad5gag + MRKAd6NSmut CV33gag + CV32NSmuttime post-homol. pre-heterol. post-heterol. point boost (T = 28) boost(T = 51) boost (T = 54) animal ID 00D088 00D099 00D240 00D088 00D09900D240 00D088 00D099 00D240 poolF 408 99 219 778 135 56 1701 1121 424poolG 47 781 844 78 363 265 228 3180 2770 poolH 49 41 87 115 50 28 97291 104 poolI 33 16 42 56 19 8 165 145 22 poolL 39 27 78 59 28 15 137815 463 poolM 44 26 78 114 28 10 219 109 21 Gag 1100 368 716 1542 237161 4460 2908 1764 DMSO 9 13 28 14 18 12 9 21 6 Vaccine MRKAd6 NSmuttime post-dose 1 post-dose 2 pre-homol. point (T = 4) (T = 8) boost (T =24) animal ID 00D065 00D116 00D159 00D065 00D116 00D159 00D065 00D11600D159 poolF 139 44 82 92 121 63 62 116 54 poolG 154 253 119 77 156 10893 165 126 poolH 1284 41 211 768 35 124 394 84 77 poolI 302 22 1174 22116 1069 134 31 561 poolL 28 16 48 35 32 21 141 113 78 poolM 1329 1007 36579 392 30 314 293 43 Gag 15 9 7 13 5 2 36 33 36 DMSO 16 4 5 9 6 4 23 178 Vaccine MRKAd6 NSmut CV32NSmut time post-homol. pre-heterol.post-heterol. point boost (T = 28) boost (T = 51) boost (T = 54) animalID 00D065 00D116 00D159 00D065 00D116 00D159 00D065 00D116 00D159 poolF44 42 23 57 85 53 313 385 261 poolG 104 59 39 44 198 48 196 764 559poolH 24 817 48 624 31 116 3758 90 925 poolI 18 133 478 84 16 362 485 512951 poolL 19 48 17 46 33 46 379 339 541 poolM 558 398 22 159 369 331278 1750 16 Gag 9 23 14 16 8 10 37 9 26 DMSO 1 9 3 23 8 6 26 9 10

Example 12 Vaccination with a ChAd Vector Comprising a TAA BreaksTolerance and Elicits a TAA-Specific T Cell Response in Monkeys

Experiments designed to determine whether chimpanzee adenoviral vectorsare sufficiently immunogenic to break the tolerance to a self-antigenand to document the utility of chimpanzee vectors for boosting an immuneresponse primed with a human adenoviral vector were performed in cohortsof four monkeys. Animals were immunized with three injection at week 0,2 and 4 of Ad5DE1 RhCEA (10^11 vp), comprising the tumor associatedantigen CEA, followed by vaccination at week 16, 18 and 20 with CV33DE1RhCEA (10^11 vp). T cell response was measured by IFNγ ELISPOT withrhesus CEA peptides.

The results reported in FIG. 34, which provide the number ofspot-forming cells per million PBMC following incubation in absence(DMSO) or in presence of rhesus CEA C and D peptides pools, establishthat an immunization protocol based on vaccination with two different Adserotypes leads to a sustained T cell response against CEA in non-humanprimates.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

What is claimed is:
 1. A recombinant chimpanzee adenoviral vectorcomprising an adenoviral genome comprising a sequence of nucleotidesthat encodes the peptide as set forth in SEQ ID NO:116, wherein saidadenoviral genome is modified in that at least one gene selected fromthe group consisting of E1, E2, E3 and E4 is functionally deleted. 2.The recombinant chimpanzee adenoviral vector according to claim 1,wherein the adenoviral genome comprises a sequence of nucleotides as setforth in SEQ ID NO:
 115. 3. The recombinant chimpanzee adenoviral vectoraccording to claim 1, wherein the vector comprises a complete deletionof its E1 genes and further wherein the vector optionally comprises acomplete deletion of its E3 genes.
 4. The recombinant chimpanzeeadenoviral vector according to claim 1, wherein the vector furthercomprises a transgene encoding at least one tumor associated antigen(TAA) operatively linked to a promoter capable of directing expressionof the transgene.
 5. The recombinant chimpanzee adenoviral vectoraccording to claim 4, wherein the at least one TAA is selected from thegroup consisting of: HER2NEU, CEA, EPCAM, PSA, PSMA, TELOMERASE, GP100,MELAN-A/MART-1, MUC-1, NY-ESO-1, SURVIVIN, STROMELYSIN 3, TYROSINASE,MAGE3, CML68, CLM66, OY-TES-1, SSX-2, SART-1, SART-2, SART-3, NY-CO-58,NY-BR-62, HKLP2, 5T4 and VEGFR2.
 6. The recombinant chimpanzeeadenoviral vector according to claim 1, wherein the vector furthercomprises a transgene encoding at least one immunogen operatively linkedto regulatory sequences which direct expression of said transgene inmammalian cells.
 7. The recombinant chimpanzee adenoviral vectoraccording to claim 6, wherein the immunogen is derived from aninfectious agent selected from the group consisting of HIV, HBV, HCV,HPV, HSV1, HSV2, SARS CoV, Plasmodium malariae, Ebola virus, West Nilevirus, Dengue virus, Influenza A, Influenza B, Mycobacteriumtuberculosis and Leishmania major.
 8. An isolated host cell comprising arecombinant chimpanzee adenoviral vector according to claim
 1. 9. Theisolated host cell of claim 8, wherein the adenoviral genome comprises asequence of nucleotides as set forth in SEQ ID NO:
 115. 10. The isolatedhost cell of claim 8, wherein the host cell is a 293 cell and whichpropagates the recombinant chimpanzee adenoviral vector.
 11. A method ofboosting an antigen-specific immune response in a mammal comprisingadministering to said mammal a sufficient amount of the recombinantchimpanzee adenoviral vector according to claim 6, which expresses atleast one antigen, and wherein the mammal was previously exposed atleast once with said at least one antigen.
 12. The method of claim 11,wherein the adenoviral genome comprises a sequence of nucleotides as setforth in SEQ ID NO:
 115. 13. The method of claim 11, wherein the vectorcomprises a complete deletion of its E1 genes and further wherein thevector optionally comprises a deletion of its E3 genes.
 14. The methodof claim 11, wherein the boosted immune response comprises theproduction of antigen-specific CD8+ T cells.
 15. The method of claim 11,wherein the immune response is a boosted immune response that isspecific for a tumor-associated antigen (TAA).
 16. The method of claim15, wherein the TAA is selected from the group consisting of: HER2NEU,CEA, EPCAM, PSA, PSMA, TELOMERASE, GP100, MELAN-A/MART-1, MUC-1,NY-ESO-1, SURVIVIN, STROMELYSIN 3, TYROSINASE, MAGE3, CML68, CLM66,OY-TES-1, SSX-2, SART-1, SART-2, SART-3, NY-CO-58, NY-BR-62, HKLP2, 5T4and VEGFR2.
 17. The method of claim 11, wherein the antigen is derivedfrom an infectious agent selected from the group consisting of HIV, HBV,HCV, HPV, HSV1, HSV2, SARS CoV, Leishmania major, Plasmodium malariae,Ebola virus, West Nile virus, Dengue virus, Influenza A, Influenza B,and Mycobacterium tuberculosis.
 18. A method of eliciting an immuneresponse in a mammal comprising administering to said mammal asufficient amount of the recombinant chimpanzee adenoviral vectoraccording to claim 6, wherein the administration of the adenoviralvector elicits an immune response.
 19. The method of claim 18, whereinthe adenoviral genome comprises the sequence of nucleotides as set forthin SEQ ID NO:
 115. 20. The method of claim 18, wherein the antigen isderived from an infectious agent selected from the group consisting ofHIV, HBV, HCV, HPV, HSV1, HSV2, SARS CoV, Leishmania major, Plasmodiummalariae, Ebola virus, West Nile virus, Dengue virus, Influenza A,Influenza B, and Mycobacterium tuberculosis.
 21. The method of claim 18,wherein the antigen is a tumor-associated antigen (TAA).
 22. The methodof claim 21, wherein the TAA is selected from the group consisting of:HER2NEU, CEA, EPCAM, PSA, PSMA, TELOMERASE, GP100, MELAN-A/MART-1,MUC-1, NY-ESO-1, SURVIVIN, STROMELYSIN 3, TYROSINASE, MAGE3, CML68,CLM66, OY-TES-1, SSX-2, SART-1, SART-2, SART-3, NY-CO-58, NY-BR-62,HKLP2, 5T4 and VEGFR2.
 23. The recombinant chimpanzee adenoviral vectorof claim 1, wherein the adenoviral genome further comprises a sequenceof nucleotides that encodes a peptide as set forth in SEQ ID NO:
 77. 24.The recombinant chimpanzee adenoviral vector according to claim 2,wherein the adenoviral genome further comprises a sequence ofnucleotides as set forth in SEQ ID NO:
 76. 25. The isolated host cell ofclaim 9, wherein the adenoviral genome further comprises a sequence ofnucleotides as set forth in SEQ ID NO:76.
 26. The method of claim 12,wherein the adenoviral genome further comprises a sequence ofnucleotides as set forth in SEQ ID NO:
 76. 27. The method of claim 20,wherein the adenoviral genome further comprises a sequence ofnucleotides as set forth in SEQ ID NO: 76.